SHIN-2 exerts potent activity against VanA-type vancomycin-resistant Enterococcus faecium in vitro by stabilizing the active site loop of serine hydroxymethyltransferase.

Novel classes of antibiotics are needed to improve the resilience of the healthcare system to antimicrobial resistance (AMR), including vancomycin resistance. vanA gene cluster is a cause of vancomycin resistance. This gene cluster is transferred and spreads vancomycin resistance from Enterococcus spp. to Staphylococcus aureus. Therefore, novel antibacterial agents are required to combat AMR, including vanA-type vancomycin resistance. Serine hydroxymethyltransferase (SHMT) is a key target of antibacterial agents. However, the specific binding mechanisms of SHMT inhibitors remain unclear. Detailed structural information will contribute to understanding these mechanisms. In this study, we found that (+)-SHIN-2, the first in vivo active inhibitor of human SHMT, is strongly bound to the Enterococcus faecium SHMT (efmSHMT). Comparison of the crystal structures of apo- and (+)-SHIN-2-boud efmSHMT revealed that (+)-SHIN-2 stabilized the active site loop of efmSHMT via hydrogen bonds, which are critical for efmSHMT inhibition. Additionally, (+)-SHIN-2 formed hydrogen bonds with serine, forming the Schiff's base with pyridoxal 5'-phosphate, which is a co-factor of SHMT. Furthermore, (+)-SHIN-2 exerted biostatic effects on vancomycin-susceptible and vanA-type vancomycin-resistant E. faecium in vitro, indicating that SHMT inhibitors do not induce cross-resistance to vanA-type vancomycin. Overall, these findings can aid in the design of novel SHMT inhibitors to combat AMR, including vancomycin resistance.

Structural and virologic mechanism of the emergence of resistance to Mpro inhibitors in SARS-CoV-2.

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (Mpro) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2WK521WT in VeroE6TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2WK521E166V), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of Mpro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to MproE166V, whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of Mpro, and compromised the enzymatic activity; the Ki values of recombinant MproE166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to MproE166V than nirmatrelvir. SARS-CoV-2WK521WT selected with 5h acquired A191T substitution in Mpro (SARS-CoV-2WK521A191T) and better replicated in the presence of 5h, than SARS-CoV-2WK521WT. However, no significant enzymatic or structural changes in MproA191T were observed. The replicability of SARS-CoV-2WK521E166V proved to be compromised compared to SARS-CoV-2WK521WT but predominated over SARS-CoV-2WK521WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2WK521A191T surpassed that of SARS-CoV-2WK521WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2's drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.

Absolute quantification of BACH1 and BACH2 transcription factors in B and plasma cells reveals their dynamic changes and unique roles.

Changes in the absolute protein amounts of transcription factors are important for regulating gene expression during cell differentiation and in responses to changes in the cellular and extracellular environment. However, few studies have focused on the absolute quantification of mammalian transcription factors. In this study, we established an absolute quantification method for the transcription factors BACH1 and BACH2, which are expressed in B cells and regulated by direct heme binding. The method used purified recombinant proteins as controls in Western blotting and was applied to mouse naïve B cells in the spleen, as well as activated B cells and plasma cells. BACH1 was present in naïve B cells at approximately half the levels of BACH2. In activated B cells, BACH1 decreased compared to naïve B cells, while BACH2 increased. In plasma cells, BACH1 increased back to the same extent as in naïve B cells, while BACH2 was not detected. Their target genes Prdm1 and Hmox1 were highly induced in plasma cells. BACH1 was found to undergo degradation with lower concentrations of heme than BACH2. Therefore, BACH1 and BACH2 are similarly abundant in B cells but differ in heme sensitivity, potentially regulating gene expression differently depending on their heme responsiveness.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Structure of a phosphodiesterase from Streptomyces sanglieri with a novel C-terminal domain.

A glycerophosphoethanolamine ethanolaminephosphodiesterase (GPE-EP) from Streptomyces sanglieri hydrolyzes glycerophosphoethanolamine to phosphoethanolamine and glycerol. The structure of GPE-EP was determined by the molecular replacement method using a search model generated with AlphaFold2. This structure includes the entire length of the mature protein and it is composed of an N-terminal domain and a novel C-terminal domain connected to a flexible linker. The N-terminal domain is the catalytic domain containing calcium ions at the catalytic site. Coordination bonds were observed between five amino acid residues and glycerol. Although the function of the C-terminal domain is currently unknown, inter-domain interactions between the N- and C-terminal domains may contribute to its relatively high thermostability.

Molecular basis of ligand recognition specificity of flavone glucosyltransferases in Nemophila menziesii.

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-ß-glucoside-4'-O-ß-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4'-O-glucosyltransferase (NmF4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (NmF4'G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4'G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4'GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4'G7GT. The substrate specificity of NmF4'GT was examined by comparing its model structure with that of NmF4'G7GT. The structure of NmF4'GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones).

Helix-based screening with structure prediction using artificial intelligence has potential for the rapid development of peptide inhibitors targeting class I viral fusion.

The rapid development of drugs against emerging and re-emerging viruses is required to prevent future pandemics. However, inhibitors usually take a long time to optimize. Here, to improve the optimization step, we used two heptad repeats (HR) in the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a model and established a screening system for peptide-based inhibitors containing an α-helix region (SPICA). SPICA can be used to identify critical amino acid regions and evaluate the inhibitory effects of peptides as decoys. We further employed an artificial intelligence structure-prediction system (AlphaFold2) for the rapid analysis of structure-activity relationships. Here, we identified that critical amino acid regions, DVDLGD (amino acids 1163-1168 in the S protein), IQKEIDRLNE (1179-1188), and NLNESLIDL (1192-1200), played a pivotal role in SARS-CoV-2 fusion. Peptides containing these critical amino acid regions efficiently blocked viral replication. We also demonstrated that AlphaFold2 could successfully predict structures similar to the reported crystal and cryo-electron microscopy structures of the post-fusion form of the SARS-CoV-2 S protein. Notably, the predicted structures of the HR1 region and the peptide-based fusion inhibitors corresponded well with the antiviral effects of each fusion inhibitor. Thus, the combination of SPICA and AlphaFold2 is a powerful tool to design viral fusion inhibitors using only the amino-acid sequence of the fusion protein.

Helical peptides with disordered regions for measles viruses provide new generalized insights into fusion inhibitors.

Despite effective vaccines, measles virus (MeV) outbreaks occur sporadically. Therefore, developing anti-MeV agents remains important for suppressing MeV infections. We previously designed peptide-based MeV fusion inhibitors, M1 and M2, that target MeV class I fusion protein (F protein). Here, we developed a novel fusion inhibitor, MEK35, that exerts potent activity against M1/M2-resistant MeV variants. Comparing MEK35 to M1 derivatives revealed that combining disordered and helical elements was essential for overcoming M1/M2 resistance. Moreover, we propose a three-step antiviral process for peptide-based fusion inhibitors: (i) disordered peptides interact with F protein; (ii) the peptides adopt a partial helical conformation and bind to F protein through hydrophobic interactions; and (iii) subsequent interactions involving the disordered region of the peptides afford a peptide-F protein with a high-affinity peptide-F protein interaction. An M1-resistant substitution blocks the second step. These results should aid the development of novel viral fusion inhibitors targeting class I F protein.

GRL-142 binds to and impairs HIV-1 integrase nuclear localization signal and potently suppresses highly INSTI-resistant HIV-1 variants.

Nuclear localization signal (NLS) of HIV-1 integrase (IN) is implicated in nuclear import of HIV-1 preintegration complex (PIC). Here, we established a multiclass drug-resistant HIV-1 variant (HIVKGD) by consecutively exposing an HIV-1 variant to various antiretroviral agents including IN strand transfer inhibitors (INSTIs). HIVKGD was extremely susceptible to a previously reported HIV-1 protease inhibitor, GRL-142, with IC50 of 130 femtomolar. When cells were exposed to HIVKGD IN-containing recombinant HIV in the presence of GRL-142, significant decrease of unintegrated 2-LTR circular cDNA was observed, suggesting that nuclear import of PIC was severely compromised by GRL-142. X-ray crystallographic analyses revealed that GRL-142 interacts with NLS's putative sequence (DQAEHLK) and sterically blocks the nuclear transport of GRL-142-bound HIVKGD's PIC. Highly INSTI-resistant HIV-1 variants isolated from heavily INSTI-experienced patients proved to be susceptible to GRL-142, suggesting that NLS-targeting agents would serve as salvage therapy agents for highly INSTI-resistant variant-harboring individuals. The data should offer a new modality to block HIV-1 infectivity and replication and shed light on developing NLS inhibitors for AIDS therapy.

The aminopeptidase LAP3 suppression accelerates myogenic differentiation via the AKT-TFE3 pathway in C2C12 myoblasts.

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin-proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X-prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl-cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine-sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched-chain amino acid levels, and enhanced mTORC2-mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation.

Little involvement of recycled-amino acids from proteasomal proteolysis in de novo protein synthesis.

Myoblast integrity is essential for skeletal muscle regeneration. Many intracellular proteins are degraded by the proteasome and converted to amino acids by aminopeptidases through the protein degradation pathway. Although we previously reported its importance for myoblast integrity, the involved mechanism remains unclear. In this study, we focused on the reusability of proteolytic products to elucidate the regulatory mechanism of protein synthesis mediated by the proteasome and aminopeptidases. Proteasome inhibition decreased protein synthesis, but recycled-amino acids derived from proteasomal proteolysis were not reused for de novo protein synthesis in C2C12 myoblasts. On the other hand, proteasome and aminopeptidase inhibition decreased intracellular ATP levels in C2C12 myoblasts. Therefore, it was indicated that amino acids produced by these proteolytic systems may be reutilized for ATP production through its metabolism, not for de novo protein synthesis. These findings suggested the proteasome and aminopeptidases are thought to be involved in protein synthesis through intracellular energy production by recycled-amino acid metabolism, thereby maintaining myoblast integrity.

Structural basis for the substrate specificity switching of lysoplasmalogen-specific phospholipase D from Thermocrispum sp. RD004668.

Lysoplasmalogen-specific phospholipase D (LyPls-PLD) hydrolyzes choline lysoplasmalogen to choline and 1-(1-alkenyl)-sn-glycero-3-phosphate. Mutation of F211 to leucine altered its substrate specificity from lysoplasmalogen to 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). Enzymes specific to lysoPAF have good potential for clinical application, and understanding the mechanism of their activity is important. The crystal structure of LyPls-PLD exhibited a TIM barrel fold assigned to glycerophosphocholine phosphodiesterase, a member of glycerophosphodiester phosphodiesterase. LyPls-PLD possesses a hydrophobic cleft for the binding of the aliphatic chain of the substrate. In the structure of the F211L mutant, Met232 and Tyr258 form a "small lid" structure that stabilizes the binding of the aliphatic chain of the substrate. In contrast, F211 may inhibit small lid formation in the wild-type structure. LysoPAF possesses a flexible aliphatic chain; therefore, a small lid is effective for stabilizing the substrate during catalytic reactions.

Autocitrullination and Changes in the Activity of Peptidylarginine Deiminase 3 Induced by High Ca2+ Concentrations.

Peptidylarginine deiminases (PADs) are enzymes that catalyze the Ca2+-dependent conversion of arginine residues into proteins to citrulline residues. Five PAD isozymes have been identified in mammals. Several studies have shown that the active-site pockets of these isozymes are formed when Ca2+ ions are properly bound. We previously characterized the structures of PAD3 in six states. Among these, we identified a "nonproductive" form of PAD3 in which the active site was disordered even though five Ca2+ ions were bound. This strange structure was probably obtained as a result of either high Ca2+ concentration (∼260 mM)-induced denaturation during the crystallization process or high Ca2+-concentration-induced autocitrullination. While autocitrullination has been reported in PAD2 and PAD4 for some time, only a single report on PAD3 has been published recently. In this study, we investigated whether PAD3 catalyzes the autocitrullination reaction and identified autocitrullination sites. In addition to the capacity of PAD3 for autocitrullination, the autocitrullination sites increased depending on the Ca2+ concentration and reaction time. These findings suggest that some of the arginine residues in the "nonproductive" form of PAD3 would be autocitrullinated. Furthermore, most of the autocitrullinated sites in PAD3 were located near the substrate-binding site. Given the high Ca2+ concentration in the crystallization condition, it is likely that Arg372 was citrullinated in the "nonproductive" PAD3 structure, the structure was slightly altered from the active form by citrulline residues, and probably inhibited Ca2+-ion binding at the proper position. Following Arg372 citrullination, PAD3 enters an inactive form; however, the Arg372-citrullinated PAD3 are considered minor components in autocitrullinated PAD3 (CitPAD3), and CitPAD3 does not significantly decrease the enzyme activity. Autocitrullination of PAD3 could not be confirmed at the low Ca2+ concentrations seen in vivo. Future experiments using cells and animals are needed to verify the effect of Ca2+ on the PAD3 structure and functions in vivo.

Serine hydroxymethyltransferase as a potential target of antibacterial agents acting synergistically with one-carbon metabolism-related inhibitors.

Serine hydroxymethyltransferase (SHMT) produces 5,10-methylenetetrahydrofolate (CH2-THF) from tetrahydrofolate with serine to glycine conversion. SHMT is a potential drug target in parasites, viruses and cancer. (+)-SHIN-1 was developed as a human SHMT inhibitor for cancer therapy. However, the potential of SHMT as an antibacterial target is unknown. Here, we show that (+)-SHIN-1 bacteriostatically inhibits the growth of Enterococcus faecium at a 50% effective concentration of 10-11 M and synergistically enhances the antibacterial activities of several nucleoside analogues. Our results, including crystal structure analysis, indicate that (+)-SHIN-1 binds tightly to E. faecium SHMT (efmSHMT). Two variable loops in SHMT are crucial for inhibitor binding, and serine binding to efmSHMT enhances the affinity of (+)-SHIN-1 by stabilising the loop structure of efmSHMT. The findings highlight the potency of SHMT as an antibacterial target and the possibility of developing SHMT inhibitors for treating bacterial, viral and parasitic infections and cancer.

The Cys-Pro motifs in the intrinsically disordered regions of the transcription factor BACH1 mediate distinct and overlapping functions upon heme binding.

The function of the transcription factor BACH1 is regulated by heme binding to multiple Cys-Pro (CP) motifs within its intrinsically disordered regions. Here, biochemical analyses were conducted to reveal the individual functional roles of the CP motifs. Our findings revealed that four CP motifs in BACH1 individually contributed to the regulation of BACH1 activity by accepting heme in a five- and six-coordination manner. The model structure around the bZip domain indicated that the CP motifs are in the N- and C-terminal heme-binding regions, which are approximately 9 nm apart, suggesting that spatial location is important for the individual function of the CP motifs. The presence of multiple CP motifs with distinct roles may ensure the multifaceted, strict regulation of BACH1 by heme.

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Lewis fucose is a key moiety for the recognition of histo-blood group antigens by GI.9 norovirus, as revealed by structural analysis.

Noroviruses have been identified as major causative agents of acute nonbacterial gastroenteritis in humans. Histo-blood group antigens (HBGAs) are thought to play a major role among the host cellular factors influencing norovirus infection. Genogroup I, genotype 9 (GI.9) is the most recently identified genotype within genogroup I, whose representative strain is the Vancouver 730 norovirus. However, the molecular interactions between host antigens and the GI.9 capsid protein have not been investigated in detail. In this study, we demonstrate that the GI.9 norovirus preferentially binds Lewis antigens over blood group A, B, and H antigens, as revealed by an HBGA binding assay using virus-like particles. We determined the crystal structures of the protruding domain of the GI.9 capsid protein in the presence or absence of Lewis antigens. Our analysis demonstrated that Lewis fucose (α1-3/4 fucose) represents a key moiety for the GI.9 protein-HBGA interaction, thus suggesting that Lewis antigens might play a critical role during norovirus infection. In addition to previously reported findings, our observations may support the future design of antiviral agents and vaccines against noroviruses.

Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2.

SARS-CoV-2-specific CD8+ T cells are scarce but detectable in unexposed healthy donors (UHDs). It remains unclear whether pre-existing human coronavirus (HCoV)-specific CD8+ T cells are converted to functionally competent T cells cross-reactive to SARS-CoV-2. Here, we identified the HLA-A24-high binding, immunodominant epitopes in SARS-CoV-2 spike region that can be recognized by seasonal coronavirus-specific CD8+ T cells from HLA-A24+ UHDs. Cross-reactive CD8+ T cells were clearly reduced in patients with hematological malignancy, who are usually immunosuppressed, compared to those in UHDs. Furthermore, we showed that CD8+ T cells in response to a selected dominant epitope display multifunctionality and cross-functionality across HCoVs in HLA-A24+ donors. Cross-reactivity of T-cell receptors isolated from them exhibited selective diversity at the single-cell level. Taken together, when stimulated well by immunodominant epitopes, selective pre-existing CD8+ T cells with high functional avidity may be cross-reactive against SARS-CoV-2.

Structural insights into the small GTPase specificity of the DOCK guanine nucleotide exchange factors.

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) regulates cytoskeletal dynamics by activating the GTPases Rac and/or Cdc42. Eleven human DOCK proteins play various important roles in developmental processes and the immune system. Of these, DOCK1-5 proteins bind to engulfment and cell motility (ELMO) proteins to perform their physiological functions. Recent structural studies have greatly enhanced our understanding of the complex and diverse mechanisms of DOCK GEF activity and GTPase recognition and its regulation by ELMO. This review is focused on gaining structural insights into the substrate specificity of the DOCK GEFs, and discuss how Rac and Cdc42 are specifically recognized by the catalytic DHR-2 and surrounding domains of DOCK or binding partners.

MK-6, a novel not-α IL-2, elicits a potent antitumor activity by improving the effector to regulatory T cell balance.

IL-2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti-immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL-2, Mutakine-6 (MK-6), with reduced IL-2Rα-binding capability. MK-6 induced comparable cell growth potential toward IL-2Rßγ-positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL-2, in vivo administration of MK-6 produced minimal adverse effects. Using CT26 and B16F10-syngeneic tumor models, we found MK-6 was highly efficacious on tumor regression. Serum albumin conjugation to MK-6 prolonged in vivo half-life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor-infiltrating leukocytes analysis revealed that albumin-fused MK-6 increased the ratio of effector CD8+ T cells to CD4+ Treg cells. These results demonstrated that MK-6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation.