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The formation of large-scale patterns through molecular self-organization is a basic principle of life. Accordingly, the engineering of protein patterns and gradients is of prime relevance for synthetic biology. As a paradigm for such pattern formation, the bacterial MinDE protein system is based on self-organization of the ATPase MinD and ATPase-activating protein MinE on lipid membranes. Min patterns can be tightly regulated by tuning physical or biochemical parameters. Among the biochemically engineerable modules, MinD's membrane targeting sequence, despite being a key regulating element, has received little attention. Here we attempt to engineer patterns by modulating the membrane affinity of MinD. Unlike the traveling waves or stationary patterns commonly observed in vitro on flat supported membranes, standing-wave oscillations emerge upon elongating MinD's membrane targeting sequence via rationally guided mutagenesis. These patterns are capable of forming gradients and thereby spatially target co-reconstituted downstream proteins, highlighting their functional potential in designing new life-like systems.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNA lys s, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNA lys , and led to a complete loss of the aminoacylation of mitochondrial tRNA lys ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3' flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3' nuclease, excises flaps at the immediate 3' end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad 3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad 3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.

The nucleus is the target of autoantibodies in many diseases, which suggests intrinsic nuclear adjuvants that confer its high autoimmunogenicity. Nucleolin (NCL) is one abundant nucleolar autoantigen in systemic lupus erythematosus (SLE) patients and, in lupus-prone mice, it elicits autoantibodies early. With purified NCL, we observed that it was a potent alarmin that activated monocytes, macrophages and dendritic cells and it was a ligand for TLR2 and TLR4. NCL released by necrotic cells also exhibited alarmin activity. The NCL alarmin activity resides in its glycine/arginine-rich (GAR/RGG) motif and can be displayed by synthetic GAR/RGG peptides. Two more GAR/RGG-containing nucleolar proteins, fibrillarin (FBRL) and GAR1, were also confirmed to be novel alarmins. Therefore, the GAR/RGG alarmin motif predicts a family of nucleolar alarmins. The apparent prevalence of nucleolar alarmins suggests their positive contribution to tissue homeostasis by inducing self-limiting tissue inflammation with autoimmunity only occurring when surveillance is broken down.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso -2,6-diaminopimelic ( meso -DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn 2+ .

Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons., Competing Interests: MB, SN, YH, DM, FH, HK, DS, EK, AK, MK, MM No competing interests declared, (© 2021, Bucher et al.)

Genome instability in cancer drives tumor heterogeneity, undermines the success of therapies, and leads to metastasis and recurrence. Condensins are conserved chromatin-binding proteins that promote genomic stability, mainly by ensuring proper condensation of chromatin and mitotic chromosome segregation. Condensin mutations are found in human tumors, but it is not known how or even if such mutations promote cancer progression. In this study, we focus on condensin II subunit CAPH2 and specific CAPH2 mutations reported to be enriched in human cancer patients, and we test how CAPH2 cancer-specific mutations may lead to condensin II complex dysfunction and contribute to genome instability. We find that R551P, R551S, and S556F mutations in CAPH2 cause genomic instability by causing DNA damage, anaphase defects, micronuclei, and chromosomal instability. DNA damage and anaphase defects are caused primarily by ataxia telangiectasia and Rad3-related-dependent telomere dysfunction, as anaphase bridges are enriched for telomeric repeat sequences. We also show that these mutations decrease the binding of CAPH2 to the ATPase subunit SMC4 as well as the rest of the condensin II complex, and decrease the amount of CAPH2 protein bound to chromatin. Thus, in vivo the R551P, R551S, and S556F cancer-specific CAPH2 mutant proteins are likely to impair condensin II complex formation, impede condensin II activity during mitosis and interphase, and promote genetic heterogeneity in cell populations that can lead to clonal outgrowth of cancer cells with highly diverse genotypes., (© 2020 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Both Colony-stimulating factor 1 receptor (CSF1R) and triggering receptor expressed on myeloid cells-2 (TREM2) are trans-membrane receptors and are expressed in the brain primarily by microglia. Mutations in these two microglia-expressed genes associated with neurodegenerative disease have recently been grouped under the term "microgliopathy". Several literatures have indicated that CSF1R and TREM2 encounters a stepwise shedding and TREM2 variants impair or accelerate the processing. However, whether CSF1R variant affects the shedding of CSF1R remains elusive. Here, plasmids containing human CSF1R or TREM2 were transiently transfected into the human embryonic kidney (HEK) 293T cells. Using Western Blot and/or ELISA assay, we demonstrated that, similar to those of TREM2, an N-terminal fragment (NTF) shedding of CSF1R ectodomain and a subsequent C-terminal fragment (CTF) of CSF1R intra-membrane were generated by a disintegrin and metalloprotease (ADAM) family member and by γ-secretase, respectively. And the shedding was inhibited by treatment with Batimastat, an ADAM inhibitor, or DAPT or compound E, a γ-secretase inhibitor. Importantly, we show that the cleaved fragments, both extracellular domain and intracellular domain of a common disease associated I794T variant, were decreased significantly. Together, our studies demonstrate a stepwise approach of human CSF1R cleavage and contribute to understand the pathogenicity of CSF1R I794T variant in adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). These studies also suggest that the cleaved ectodomain fragment released from CSF1R may be proposed as a diagnostic biomarker for ALSP., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Substitution for aspartic acid (D) by glycine (G) at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. Here, we report cryo-electron microscopy structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations that differ primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer-effectively increasing the number of functional spikes and enhancing infectivity-and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro . In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.

Missense variants are present amongst the healthy population, but some of them are causative of human diseases. A classification of variants associated with "healthy" or "diseased" states is therefore not always straightforward. A deeper understanding of the nature of missense variants in health and disease, the cellular processes they may affect, and the general molecular principles which underlie these differences is essential to offer mechanistic explanations of the true impact of pathogenic variants. Here, we have formalised a statistical framework which enables robust probabilistic quantification of variant enrichment across full-length proteins, their domains, and 3D structure-defined regions. Using this framework, we validate and extend previously reported trends of variant enrichment in different protein structural regions (surface/core/interface). By examining the association of variant enrichment with available functional pathways and transcriptomic and proteomic (protein half-life, thermal stability, abundance) data, we have mined a rich set of molecular features which distinguish between pathogenic and population variants: Pathogenic variants mainly affect proteins involved in cell proliferation and nucleotide processing and are enriched in more abundant proteins. Additionally, rare population variants display features closer to common than pathogenic variants. We validate the association between these molecular features and variant pathogenicity by comparing against existing in silico variant impact annotations. This study provides molecular details into how different proteins exhibit resilience and/or sensitivity towards missense variants and provides the rationale to prioritise variant-enriched proteins and protein domains for therapeutic targeting and development. The ZoomVar database, which we created for this study, is available at fraternalilab.kcl.ac.uk/ZoomVar. It allows users to programmatically annotate missense variants with protein structural information and to calculate variant enrichment in different protein structural regions., Competing Interests: The authors have declared that no competing interests exist.

Background: The G-protein-coupled receptor GPR55 has been implicated in multiple biological activities, which has fuelled interest in its functional targeting. Its controversial pharmacology and often species-dependent regulation have impacted upon the potential translation of preclinical data involving GPR55., Results: With the aim to identify novel GPR55 regulators, we have investigated lysophosphatidylinositol (LPI)-induced GPR55-mediated signal transduction. The expression system for wild-type and mutated GPR55 was HeLa cells silenced for their endogenous receptor by stable expression of a short-hairpin RNA specific for GPR55 5'-UTR, which allowed definition of the requirement of GPR55 Lys 80 for LPI-induced MAPK activation and receptor internalisation. In RAW264.7 macrophages, GPR55 pathways were investigated by Gpr55 silencing using small-interfering RNAs, which demonstrated that LPI increased intracellular Ca 2+ levels and induced actin filopodium formation through GPR55 activation. Furthermore, the LPI/GPR55 axis was shown to have an active role in osteoclastogenesis of precursor RAW264.7 cells induced by 'receptor-activator of nuclear factor kappa-β ligand' (RANKL). Indeed, this differentiation into mature osteoclasts was associated with a 14-fold increase in Gpr55 mRNA levels. Moreover, GPR55 silencing and antagonism impaired RANKL-induced transcription of the osteoclastogenesis markers: 'nuclear factor of activated T-cells, cytoplasmic 1', matrix metalloproteinase-9, cathepsin-K, tartrate-resistant acid phosphatase, and the calcitonin receptor, as evaluated by real-time PCR. Phage display was previously used to identify peptides that bind to GPR55. Here, the GPR55-specific peptide-P1 strongly inhibited osteoclast maturation of RAW264.7 macrophages, confirming its activity as a blocker of GPR55-mediated functions. Although osteoclast syncytium formation was not affected by pharmacological regulation of GPR55, osteoclast activity was dependent on GPR55 signalling, as shown with resorption assays on bone slices, where LPI stimulated and GPR55 antagonists inhibited bone erosion., Conclusions: Our data indicate that GPR55 represents a target for development of novel therapeutic approaches for treatment of pathological conditions caused by osteoclast-exacerbated bone degradation, such as in osteoporosis or during establishment of bone metastases. Video abstract.

Cells probe their surrounding matrix for attachment sites via integrins that are internalized by endocytosis. We find that SH3BP4 regulates integrin surface expression in a signaling-dependent manner via clathrin-coated pits (CCPs). Dephosphorylated SH3BP4 at S246 is efficiently recruited to CCPs, while upon Akt phosphorylation, SH3BP4 is sequestered by 14-3-3 adaptors and excluded from CCPs. In the absence of Akt activity, SH3BP4 binds GIPC1 and targets neuropilin-1 and α5/β1-integrin for endocytosis, leading to inhibition of cell spreading. Similarly, chemorepellent semaphorin-3a binds neuropilin-1 to activate PTEN, which antagonizes Akt and thus recruits SH3BP4 to CCPs to internalize both receptors and induce cell contraction. In PTEN mutant non-small cell lung cancer cells with high Akt activity, expression of non-phosphorylatable active SH3BP4-S246A restores semaphorin-3a induced cell contraction. Thus, SH3BP4 links Akt signaling to endocytosis of NRP1 and α5/β1-integrins to modulate cell-matrix interactions in response to intrinsic and extrinsic cues., Competing Interests: Declaration of interests The authors declare no competing interests. G.D. is a member of the editorial board of Developmental Cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Glucose oxidase (Gox) is a biocatalyst that is widely applied in the food industry, as well as other biotechnological industries. However, the industrial application of Gox is hampered by its low thermostability and activity. Here, we aimed to improve the thermostability of GoxM4 from Aspergillus niger without reducing its activity due to the activity-stability trade-off. A simple and effective approach combining enzyme activity and structure stability was adopted to evaluate the thermostability of GoxM4 and its mutants. After four rounds of computer-aided rational design, the best mutant, GoxM8, was obtained. The melting temperature (T m ) of GoxM8 was increased by 9 °C compared with GoxM4. The catalytic efficiency of GoxM8 was similar to GoxM4, suggesting that the enzyme activity-stability trade-off was counteracted. To explore its mechanism, we performed molecular dynamics simulations of GoxM4 and its mutants. Our findings provided a typical example for researching the enzyme activity-stability trade-off., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

TDP-43 proteinopathy is implicated in the neurodegenerative diseases, ALS and FTLD-TDP. Metal ion dyshomeostasis is observed in neurodegenerative diseases including ALS. Previously, mice expressing A315T familial ALS TDP-43 mutant showed elevated spinal cord Zn 2+ levels. Recently, Zn 2+ was observed to modulate the in vitro amyloid-like aggregation of the TDP-43's RRM12 domains. As a systematic knowledge of the TDP-43's interaction with Zn 2+ is lacking, we in silico predicted potential Zn 2+ binding sites in TDP-43 and estimated their relative solvent accessibilities. Zn 2+ binding sites were predicted in the TDP-43's N-terminal domain, in the linker region between RRM1 and RRM2 domain, within RRM2 domain and at the junction of the RRM2 and C-terminal domain (CTD), but none in the 311-360 region of CTD. Furthermore, we found that Zn 2+ promotes the in vitro thioflavin-T-positive aggregations of C-terminal fragments (CTFs) termed TDP-43 2C and TDP-43 2C -A315T that encompass the RRM2 and CTD domains. Also, while the Alexa-fluor fluorescently labelled TDP-43 2C and TDP-43 2C -A315T proteins manifested liquid-like spherical droplets, Zn 2+ caused a solid-like phase separation that was not ameliorated even by carboxymethylation of the free cysteines thereby implicating the other Zn 2+ -binding residues. The observed Zn 2+ -promoted TDP-43 CTF's solid-like phase separation can be relevant to the Zn 2+ dyshomeostasis in ALS and FTLD-TDP., Competing Interests: Declaration of competing interest Authors declare that no competing interests exist., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The forward (k on ) and reverse (k off ) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the k on s and k off s of four dopamine D 2 receptor (D 2 R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S193 5.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar k off s for the two 5-OH-DPAT enantiomers at wild-type (WT) D 2 R, both being slower than the k off s of DA and p-tyramine. Conversely, the k on of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S193 5.42 A mutation lowered the k on of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic K d s derived from the k off and k on estimates correlated well with EC 50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D 2 R agonist candidate drugs.

In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting "hot regions," i.e., amino acid substitutions therein frequently increase the affinities, is desirable for straightforward discovery of valuable mutants. We here report two "designed" site-directed mutagenesis (A and B) targeted the N-terminal 1-10 positions of the V H framework region 1 that successfully improved an anti-cortisol single-chain Fv fragment (K a , 3.6 × 10 8  M -1 ). Mutagenesis A substituted the amino acids at the position 1-3, 5-7, 9 and 10 with a limited set of substitutions to generate only 1,536 different members, while mutagenesis B inserted 1-6 random residues between the positions 6 and 7. Screening the resulting bacterial libraries as scFv-phage clones with a clonal array profiling system provided 21 genetically unique scFv mutants showing 17-31-fold increased affinity with > 10 9  M -1 K a values. Among the mutants selected from the library A and B, scFv mA#18 (with five-residue substitutions) and mB 1-3 #130 (with a single residue insertion) showed the greatest K a value, 1.1 × 10 10  M -1 .

Introduction: Peptides can be rationally designed as non-covalent inhibitors for molecularly targeted therapy. However, it remains challenging to efficiently deliver the peptides into the targeted cells, which often severely affects their therapeutic efficiency., Methods: Herein, we created a novel non-covalent peptide inhibitor against nuclear export factor CRM1 by a structure-guided drug design method and targetedly delivered the peptide into cancer cells by a nanoparticle-mediated gene expression system for use as a cancer therapy., Results: The nuclear export signal (NES)-optimized CRM1 peptide inhibitor colocalized with CRM1 to the nuclear envelope and inhibited nuclear export in cancer cell lines in vitro. The crystal structures of the inhibitors complexed with CRM1 were solved. In contrast to the covalent inhibitors, the peptides were similarly effective against cells harboring the CRM1 C528S mutation. Moreover, a plasmid encoding the peptides was delivered by a iRGD-modified nanoparticle to efficiently target and transfect the cancer cells in vivo after intravenous administration. The peptides could be selectively expressed in the tumor, resulting in the efficient inhibition of subcutaneous melanoma xenografts without obvious systemic toxicity., Discussion: This work provides an effective strategy to design peptide-based molecularly targeted therapeutics, which could lead to the development of future targeted therapy., Competing Interests: QS, YL, MS, MG, and MX have applied patents on the designed peptides in this study. The authors report no other conflicts of interest in this work., (© 2021 Sui et al.)

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca 2+ -dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy-skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2 . Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca 2+ , although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca 2+ ]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca 2+ ]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca 2+ -dependent regulation of actin-myosin interactions.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1 G71A , the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.

R2TP is a well-conserved molecular chaperone complex, composed of Pontin, Reptin, RPAP3, and PIH1D, in eukaryotes. Recent studies have suggested an involvement of R2TP in cancer development. However, it remains unclear if it is related to the development of oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer. Here, we identify and investigate the function of R2TP in OSCC development. Immunohistochemical analysis reveals that all of the R2TP components are strongly expressed in normal oral epithelia and OSCC tissues, where actively proliferating cells are abundant. Co-immunoprecipitation assay identifies that R2TP components form a protein complex in OSCC-derived HSC4-cells. Knockdown experiments show that all R2TP components, except for RPAP3, are required for the cell proliferation and migration of HSC-4 cells. Furthermore, we reveal that Pontin contributes to a gain-of-function (GOF) activity of mutp53-R248Q in HSC-4 cells by regulating phosphorylation levels of mutp53 at Ser15 and Ser46. To our knowledge, this study is the first to report the functional involvement of R2TP and its components in the malignant characteristics of OSCC cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Pannexin 1 (Panx1) has been implicated in a plethora of physiological and pathophysiological processes. It is one of the major ATP release channels in many cell types. Extracellular ATP, activates purinergic P2X and P2Y receptors, triggering several signaling cascades. A disease-associated mutation, Arg-217-His (R217H) in the 3rd transmembrane domain of Panx1 attenuates channel functions through an unknown mechanism. Since carboxyl terminus (CT) gates the channel, we hypothesized that R217 interacts with the CT, and this interaction is required for optimum channel activities. R217H mutation though reduced the currents in the full-length channel, did not affect CT-truncated Panx1-Δ386. Also, compared to the wild-type, Panx1-R217H expressing cells showed lesser cell death when activated through P2X7 receptor. However, cell death in Panx1-R217H-Δ386 and Panx1-Δ386 expressing cells were similar. The mutation is ineffective unless the channel has an intact CT. Based on our results we propose that R217H mutation perturbs the conformational flexibility of CT, leading to channel dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.

Mutant p53(s) are widely considered as oncogenes and promote several gain-of-function oncogenic activities. p53 mutations correlate with higher rates of metastasis and poor survival; therefore, it is paramount to inhibit mutant p53 protein either directly or indirectly. Although some compounds have been developed, none of them have achieved a desirable level of specificity. Some of these compounds only targeted specific mutations. In search of less-toxic compounds, we tested plant-derived compounds on mutant p53 triple-negative breast cancer cell lines. Here, we show that the compounds tested reduced the protein levels of one of the more frequent oncogenic p53 mutants (R249S; hot spot mutation), and its important targets that promote invasion and metastasis, including GMPS and IMPDH1. All compounds tested perturbed the invasion potential of the breast cancer cell line. These compounds downregulated several nucleotide metabolism genes (NMGs) which are essential for cell cycle progression. We observed S-phase arrest correlating to reduced cell proliferation and increased replication stress. Moreover, we also show a reduction of key ETS transcription family members including ETS2, ETS1, ETV1, and ETV4, which are involved in invasion and metastasis. We propose that these compounds may inhibit invasion by interfering with multiple pathways. Our findings exemplify that these tested compounds could inhibit invasion and cell growth in TNBC in a nucleotide-dependent manner.

We has identified a founder homozygous E3_E4 del: 2870 bp deletion + 9 bp insertion in AGT gene encoding angiotensinogen responsible for autosomal recessive renal tubular dysgenesis (ARRTD) with nearly-fatal outcome. High-dose hydrocortisone therapy successfully rescued one patient with an increased serum Angiotensinogen (AGT), Ang I, and Ang II levels. The pathogenesis of ARRTD caused by this AGT mutation and the potential therapeutic effect of hydrocortisone were examined by in vitro functional studies. The expression of this truncated AGT protein was relatively low with a dose-dependent manner. This truncated mutation diminished the interaction between mutant AGT and renin. The truncated AGT also altered the glucocorticoid receptor (GR)-dependent transactivation, indicating that AGT may affect the development of proximal convoluted tubule by alteration of glucocorticoid-dependent transactivation. In hepatocytes, hydrocortisone increased the AGT level by accentuating the stability of mutant AGT and increasing its binding with renin. Therefore, hydrocortisone may exert the therapeutic effect through the enhanced stability and interaction with renin of truncated AGT in patients carrying this AGT mutation.

Point mutations within the DNA-binding domain of the TP53 gene occur in a significant percentage of human cancer, leading to cellular accumulation of highly stabilized mutant p53 proteins (mutp53) with tumor-promoting properties. Depletion of mutp53, through inducing either autophagic or proteasomal degradation, is an attractive strategy for the therapy of p53-mutated cancer, but the currently-known degradation inducers, almost exclusively small molecules, are inadequate. Here we show that pH-responsive zeolitic imidazolate framework-8 (ZIF-8) offers a novel solution to mutp53 degradation. ZIF-8 facilitated ubiquitination-mediated and glutathionylation-dependent proteasomal degradation of all of the nine mutp53 we tested, including six hot-spot mutp53, but not the wild-type p53 protein. Sustained elevation of intracellular Zn ++ level, resulted from decomposition of the internalized ZIF-8 in the acidic endosomes, decreased the intracellular reduced glutathione (GSH): oxidized glutathione (GSSG) ratio and was essential for mutp53 glutathionylation and degradation. ZIF-8 modified with an Z1-RGD peptide, exhibiting enhanced cellular internalization and improved decomposition behavior, preferentially killed mutp53-expressing cancer cells and demonstrated remarkable therapeutic efficacy in a p53 S241F ES-2 ovarian cancer model as well as in a p53 Y220C patient-derived xenograft (PDX) breast cancer model. The ability to induce wide-spectrum mutp53 degradation gives ZIF-8 a clear advantage over other degradation-inducers, and engineered nanomaterials may be promising alternatives to small molecules for the development of mutp53-targeting drugs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The 14 kDa histidine triad nucleotide-binding protein 1 (HINT1) is critical to maintain the normal function of motor neurons. Thus, a series of human HINT1 mutants cause autosomal recessive axonal neuropathy with neuromyotonia. HINT1 establishes a series of regulatory interactions with signaling proteins, some of which are enriched in motor neurons, such as the type 1 sigma receptor or intracellular domain (ICD) of transmembrane teneurin 1, both of which are also implicated in motor disturbances. In a previous study, we reported the capacity of HINT1 to remove the small ubiquitin-like modifier (SUMO) from a series of substrates and the influence of HINT1 mutants on this activity. We now report how human HINT1 mutations affect the interaction of HINT1 with the regulator of its SUMOylase activity, calcium-activated calmodulin, and its substrate SUMO. Moreover, HINT1 mutants exhibited anomalous interactions with G protein coupled receptors, such as the mu-opioid, and with glutamate N-methyl-D-aspartate receptors as well. Additionally, these HINT1 mutants showed impaired associations with transcriptional regulators such as the regulator of G protein signaling Z2 protein and the cleaved N-terminal ICD of teneurin 1. Thus, the altered enzymatic activity of human HINT1 mutants and their anomalous interactions with partner proteins may disrupt signaling pathways essential to the normal function of human motor neurons.

This study aimed to investigate four of the eight PFN-1 mutations that are located near the actin-binding domain and determine the structural changes due to each mutant and unravel how these mutations alter protein structural behavior. Swapaa's command in UCSF chimera for generating mutations, FTMAP were employed and the data was analyzed by RMSD, RMSF graphs, Rg, hydrogen bonding analysis, and RRdisMaps utilizing Autodock4 and GROMACS. The functional changes and virtual screening, structural dynamics, and chemical bonding behavior changes, molecular docking simulation with two current FDA-approved drugs for ALS were investigated. The highest reduction and increase in Rg were found to exist in the G117V and M113T mutants, respectively. The RMSF data consistently shows changes nearby to this site. The in silico data described indicate that each of the mutations is capable of altering the structure of PFN-1 in vivo. The potential effect of riluzole and edaravone two FDA approved drugs for ALS, impacting the structural deviations and stabilization of the mutant PFN-1 is evaluated using in silico tools. Overall, the analysis of data collected reveals structural changes of mutant PFN-1 protein that may explain the neurotoxicity and the reason(s) for possible loss and gain of function of PFN-1 in the neurotoxic model of ALS.

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gα s protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, β-arrestin recruitment, and/or coupling to Gα s , establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.

β-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the β-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.

Aims: Mutations in PIK3CA, which encodes p110α subunit of PI3K class IA enzymes, are highly frequent in breast cancer. Here, we aimed to probe mutations in exon 9 of PIK3CA and computationally simulate their function., Materials and Methods: PCR/HRM and PCR/sequencing were used for mutation detection in 40 breast cancer specimens. The identified mutations were queried via in silico algorithms to check the pathogenicity. The molecular dynamics (MD) simulations were utilized to assess the function of mutant proteins., Key Findings: Three samples were found to harbor at least one of the E542K, E545K and L551Q mutations of which L551Q has not been reported previously. All mutations were confirmed to be pathogenic and MD simulations revealed their impact on protein function and regulation. The novel L551Q mutant dynamics was similar to that of previously found carcinogenic mutants, E542K and E545K. A functional role for the helical domain was also suggested by which the inhibitory signal of p85α is conducted to kinase domain via helical domain. Helical domain mutations lead to impairment of kinase domain allosteric regulation. Interestingly, our results show that p110α substrate binding pocket of kinase domain in mutants may have differential affinity for enzyme substrates, including anit-p110α drugs., Significance: The novel p110α L551Q mutation could have carcinogenic feature similar to previously known helical domain mutations., (Copyright © 2020. Published by Elsevier Inc.)

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

Aquaporin-4 is a transmembrane water channel protein, the C-terminal domain of which is facing the cytosol. In the process of investigating the role of the C-terminal domain of aquaporin-4 with regard to intracellular trafficking, we observed that a derivative of aquaporin-4, in which the C-terminal 53 amino acids had been removed (Δ271-323), was localized to intracellular compartments, including the endoplasmic reticulum, but was not expressed on the plasma membranes. This was determined by immunofluorescence staining and labeling of the cells with monoclonal antibody specifically recognizing the extracellular domain of aquaporin-4, followed by confocal microscopy and flow cytometry. Deletion of additional amino acids in the C-terminal domain of aquaporin-4 led to its redistribution to the plasma membrane. This suggests that the effect of the 53-amino acid deletion on the subcellular localization of aquaporin-4 could be attributed to the formation of a signal at the C terminus that retained aquaporin-4 in intracellular compartments, rather than the loss of a signal required for plasma membrane targeting. Substitution of the lysine at position 268 with alanine could rescue the Δ271-323-associated retention in the cytosol, suggesting that the C-terminal sequence of the mutant served as a signal similar to a di-lysine motif., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Caveolae are specialized domains of the vertebrate cell surface with a well-defined morphology and crucial roles in cell migration and mechanoprotection. Unique compositions of proteins and lipids determine membrane architectures. The precise caveolar lipid profile and the roles of the major caveolar structural proteins, caveolins and cavins, in selectively sorting lipids have not been defined. Here, we used quantitative nanoscale lipid mapping together with molecular dynamic simulations to define the caveolar lipid profile. We show that caveolin-1 (CAV1) and cavin1 individually sort distinct plasma membrane lipids. Intact caveolar structures composed of both CAV1 and cavin1 further generate a unique lipid nano-environment. The caveolar lipid sorting capability includes selectivities for lipid headgroups and acyl chains. Because lipid headgroup metabolism and acyl chain remodeling are tightly regulated, this selective lipid sorting may allow caveolae to act as transit hubs to direct communications among lipid metabolism, vesicular trafficking, and signaling., (© 2021 Zhou et al.)

Key Message: We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

The molecular mechanisms underlying neurodevelopmental disorders (NDDs) remain unclear. We previously identified Down syndrome cell adhesion molecule like 1 (Dscaml1) as a responsible gene for Ihara epileptic rat (IER), a rat model for human NDDs with epilepsy. However, the relationship between NDDs and DSCAML1 in humans is still elusive. In this study, we screened databases of autism spectrum disorders (ASD), intellectual disability (ID)/developmental disorders (DD) and schizophrenia for genomic mutations in human DSCAML1. We then performed in silico analyses to estimate the potential damage to the mutated DSCAML1 proteins and chose three representative mutations (DSCAML1 C729R , DSCAML1 R1685* and DSCAML1 K2108Nfs*37 ), which lacked a cysteine residue in the seventh Ig domain, the intracellular region and the C-terminal PDZ-binding motif, respectively. In overexpression experiments in a cell line, DSCAML1 C729R lost its mature N-glycosylation, whereas DSCAML1 K2108Nfs*37 was abnormally degraded via proteasome-dependent protein degradation. Furthermore, in primary hippocampal neurons, the ability of the wild-type DSCAML1 to regulate the number of synapses was lost with all mutant proteins. These results provide insight into understanding the roles of the domains in the DSCAML1 protein and further suggest that these mutations cause functional changes, albeit through different mechanisms, that likely affect the pathophysiology of NDDs., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic 1 -has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses 2 . To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components., (© 2021 Rüthnick et al.)

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.

The frameshift mutants K192S fs*7 and R153S fs*41 , of the polyglutamine tract-binding protein 1 (PQBP-1), are stable intrinsically disordered proteins (IDPs). They are each associated with the severe cognitive disorder known as the Renpenning syndrome, a form of X-linked intellectual disability (XLID). Relative to the monomeric wild-type protein, these mutants are dimeric, contain more folded contents, and have higher thermal stabilities. Comparisons can be drawn to the toxic oligomerisation in the "conformational diseases", which collectively describe medical conditions involving a substantial protein structural transition in the pathogenic mechanism. At the molecular level, the end state of these diseases is often cytotoxic protein aggregation. The conformational disease proteins contain varying extents of intrinsic disorder, and the consensus pathogenesis includes an early oligomer formation. We reviewed the experimental characterisation of the toxic oligomers in representative cases. PQBP-1 mutant dimerisation was then compared to the oligomerisation of the conformational disease proteins. The PQBP-1 mutants are unique in behaving as stable soluble dimers, which do not further develop into higher oligomers or aggregates. The toxicity of the PQBP-1 mutant dimers lies in the native functions (in transcription regulation and possibly, RNA splicing) being compromised, rather than proceeding to aggregation. Other examples of stable IDP dimers were discussed and we speculated on the roles of IDP dimerisation in protein evolution.

Upon starvation, cells rewire their metabolism, switching from glucose-based metabolism to mitochondrial oxidation of fatty acids, which require the transfer of FAs from lipid droplets (LDs) to mitochondria at mitochondria-LD membrane contact sites (MCSs). However, factors responsible for FA transfer at these MCSs remain uncharacterized. Here, we demonstrate that vacuolar protein sorting-associated protein 13D (VPS13D), loss-of-function mutations of which cause spastic ataxia, coordinates FA trafficking in conjunction with the endosomal sorting complex required for transport (ESCRT) protein tumor susceptibility 101 (TSG101). The VPS13 adaptor-binding domain of VPS13D and TSG101 directly remodels LD membranes in a cooperative manner. The lipid transfer domain of human VPS13D binds glycerophospholipids and FAs in vitro. Depletion of VPS13D, TSG101, or ESCRT-III proteins inhibits FA trafficking from LDs to mitochondria. Our findings suggest that VPS13D mediates the ESCRT-dependent remodeling of LD membranes to facilitate FA transfer at mitochondria-LD contacts.

Synonymous mutation of the N-terminal coding sequence (NCS) has been used to regulate gene expression. We here developed a statistical model to predict the effect of the NCSs on protein expression in Bacillus subtilis WB600. First, a synonymous mutation was performed within the first 10 residues of a superfolder green fluorescent protein to generate a library of 172 NCS synonymous mutants with different expression levels. A prediction model was then developed, which adopted G/C frequency at the third position of each codon and minimum free energy of mRNA as the independent variables, using multiple regression analysis between the 11 sequence parameters of the NCS and their fluorescence intensities. By designing the NCS of the 10 signal peptides de novo according to the model, the extracellular yield of B. subtilis pullulanase fused to each signal peptide was up-regulated by up to 515% or down-regulated by at most 79%. This work provided a candidate tool for fine-tuning gene expression or enzyme production in B. subtilis .