Curvature-sensing mechanisms assist proteins in executing particular actions on various membrane organelles. Here, we investigate the functional specificity of curvature-sensing amphipathic motifs in Caenorhabditis elegans through the study of endophilin, an endocytic protein for synaptic vesicle recycling. We generate chimeric endophilin proteins by replacing the endophilin amphipathic motif H0 with other curvature-sensing amphipathic motifs. We find that the role of amphipathic motifs cannot simply be extrapolated from the identity of their parental proteins. For example, the amphipathic motif of the nuclear pore complex protein NUP133 functionally replaces the synaptic role of endophilin H0. Interestingly, non-functional endophilin chimeras have similar defects-producing fewer synaptic vesicles but more endosomes-and this indicates that the curvature-sensing motifs in these chimeras have a common deficiency for reforming synaptic vesicles. Finally, we convert non-functional endophilin chimeras into functional proteins by changing the cationic property of amphipathic motifs, successfully reprogramming the functional specificity of curvature-sensing motifs in vivo.

In recent years, it has been established that atherosclerosis is an autoimmune disease. However, little is currently known about the role of FcγRIIA in atherosclerosis. Herein, we sought to investigate the relationship between FcγRIIA genotypes and the effectiveness of different IgG subclasses in treating atherosclerosis. We constructed and produced different subtypes of IgG and Fc-engineered antibodies. In vitro, we observed the effect of different subtypes of IgG and Fc-engineered antibodies on the differentiation of CD14+ monocytes from patients or healthy individuals. In vivo, Apoe-/- mice were fed a high-fat diet (HFD) for 20 weeks and administered injections of different CVI-IgG subclasses or Fc-engineered antibodies. Flow cytometry was used to assess the polarization of monocytes and macrophages. Although CVI-IgG4 reduced the release of MCP-1 compared to the other subtypes, IgG4 did not yield an anti-inflammatory effect by induction of human monocyte and macrophage differentiation in vitro. Furthermore, genetic polymorphisms of FcγRIIA were not associated with different CVI-IgG subclasses during the treatment of atherosclerosis. In vivo, CVI-IgG1 decreased Ly6Chigh monocyte differentiation and promoted M2 macrophage polarization. We also found that the secretion of IL-10 was upregulated in the CVI-IgG1-treated group, whereas V11 and GAALIE exerted no significant effect. These findings highlight that IgG1 is the optimal subtype for treating atherosclerosis, and CVI-IgG1 can induce monocyte/macrophage polarization. Overall, these results have important implications for the development of therapeutic antibodies.

CDK11, a member of the cyclin-dependent kinase family, has been implicated in a diverse array of functions including transcription, RNA processing, sister chromatid cohesion, spindle assembly, centriole duplication and apoptosis. Despite its involvement in many essential functions, little is known about the requirements for CDK11 and its partner Cyclin L in a developing multicellular organism. Here we investigate the function of CDK11 and Cyclin L during development of the nematode Caenorhabditis elegans. Worms express two CDK11 proteins encoded by distinct loci: CDK-11.1 is essential for normal male and female fertility and is broadly expressed in the nuclei of somatic and germ line cells, while CDK-11.2 is nonessential and is enriched in hermaphrodite germ line nuclei beginning in mid pachytene. Hermaphrodites lacking CDK-11.1 develop normally but possess fewer mature sperm and oocytes and do not fully activate the RAS-ERK pathway that is required for oocyte production in response to environmental cues. Most of the sperm and eggs that are produced in cdk-11.1 null animals appear to complete development normally but fail to engage in sperm-oocyte signaling suggesting that CDK-11.1 is needed at multiple points in gametogenesis. Finally, we find that CDK-11.1 and CDK-11.2 function redundantly during embryonic and postembryonic development and likely do so in association with Cyclin L. Our results thus define multiple requirements for CDK-11-Cyclin L during animal development.

Gibbons have experienced extensive karyotype rearrangements during evolution and represent an ideal model for studying the underlying molecular mechanism of evolutionary chromosomal rearrangements. It is anticipated that the cloning and sequence characterization of evolutionary chromosomal breakpoints will provide vital insights into the molecular force that has driven such a radical karyotype reshuffle in gibbons. We constructed and characterized a high-quality fosmid library of the white-cheeked gibbon (Nomascus leucogenys) containing 192,000 non- redundant clones with an average insert size of 38 kb and 2.5-fold genome coverage. By end sequencing of 100 randomly selected fosmid clones, we generated 196 sequence tags for the library. These end-sequenced fosmid clones were then mapped onto the chromosomes of the white-cheeked gibbon by fluorescence in situ hybridization, and no spurious chimeric clone was detected. BLAST search against the human genome showed a good correlation between the number of hit clones and the number of chromosomes, an indication of unbiased chromosomal distribution of the fosmid library. The chromosomal distribution of the mapped clones is also consistent with the BLAST search result against human and white-cheeked gibbon genomes. The fosmid library and the mapped clones will serve as a valuable resource for further studying gibbons' chromosomal rearrangements and the underlying molecular mechanism as well as for comparative genomic study in the lesser apes.

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.

Successful muscle regeneration following injury is essential for functional homeostasis of skeletal muscles. Krüppel-like factor 15 (KLF15) is a metabolic transcriptional regulator in the muscles. However, little is known regarding its function in muscle regeneration. Here, we examined microarray datasets from the Gene Expression Omnibus database, which indicated downregulated KLF15 in muscles from patients with various muscle diseases. Additionally, we found that Klf15 knockout (Klf15KO) impaired muscle regeneration following injury in mice. Furthermore, KLF15 expression was robustly induced during myoblast differentiation. Myoblasts with KLF15 deficiency showed a marked reduction in their fusion capacity. Unbiased transcriptome analysis of muscles on day 7 postinjury revealed downregulated genes involved in cell differentiation and metabolic processes in Klf15KO muscles. The FK506-binding protein 51 (FKBP5), a positive regulator of myoblast differentiation, was ranked as one of the most strongly downregulated genes in the Klf15KO group. A mechanistic search revealed that KLF15 binds directly to the promoter region of FKBP5 and activates FKBP5 expression. Local delivery of FKBP5 rescued the impaired muscle regeneration in Klf15KO mice. Our findings reveal a positive regulatory role of KLF15 in myoblast differentiation and muscle regeneration by activating FKBP5 expression. KLF15 signaling may be a novel therapeutic target for muscle disorders associated with injuries or diseases.

Cardiac fibrosis is pathological damage associated with nearly all forms of heart disease. AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme. Emerging evidences indicate that AMPK plays an important role in cardiac fibrosis and cell proliferation. However, less is known about the detailed mechanism of AMPK activation on cardiac fibrosis. In this study, we found the AMPK activation improved the impaired cardiac function of cardiac fibrosis rats and decreased interstitial fibrosis. Further results indicated AMPK activation promoted p21 and p27 and inhibited CDK2 and cyclin E protein expressions both in vivo and in vitro. Moreover, AMPK activation repressed downstream transcription factor hepatocyte nuclear factor 4 alpha (HNF-4α) expression and decreased the binding of HNF-4α to TGF-β1 promoters, which eventually resulted in TGF-β1 downregulation and miR-29 family upregulation. Furthermore, miR-29, in turn, inhibited the progression of cardiac fibrosis through suppressing its target CDK2. Taken together, activation of AMPK, on the one hand, upregulated p21 and p27 expression, further inhibited CDK2 and cyclin E complex, and finally suppressed the progression of cardiac fibrosis, and, on the other hand, repressed HNF-4α expression, further downregulated the activity of TGF-β1 promoter, promoted miR-29 expression, and finally prevented the development of cardiac fibrosis.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

MYC and HIF1α are among the most important oncoproteins whose pharmacologic inhibition has been challenging for the diverse mechanisms driving their abnormal expression and because of the challenge in blocking protein-DNA interactions. Surprisingly, we found that MYC and HIF1α proteins in echinomycin-treated cells were degraded through proteasome dependent pathways, respectively by the β-TrCP- or VHL-dependent mechanisms. The degradation is induced in a variety of cancer types, including those with mutations in the p53 tumor and LKB tumor suppressors and the KRAS oncogene. Consistent with inhibition of MYC and HIF1α, administration of echinomycin inhibited growth of lung adenocarcinoma xenograft and a syngeneic lymphoma model in mice. Furthermore, echinomycin efficiently induced regression of syngeneic mouse lymphoma driven by MYC over-expression. Our data demonstrated a new mechanism by which echinomycin simultaneously targets MYC and HIF1α for degradation to inhibit growth of lung cancer and lymphoma. Given the broad impact of β-TrCP or VHL in stability of oncogenic proteins, echinomycin may emerge as a non-PROTAC (proteolysis targeting chimera) degrader of oncogenic proteins.

Circular RNAs (circRNAs) are implicated in the chemoresistance of human cancers. However, the functions of circRNA PR/SET domain 2 (circPRDM2) in the resistance of osteosarcoma (OS) to doxorubicin (DXR) are unknown.

Shank proteins, one of the principal scaffolds in the postsynaptic density (PSD) of the glutamatergic synapses, have been associated with autism spectrum disorders and neuropsychiatric diseases. However, it is not known whether different Shank family proteins have distinct functions in regulating synaptic transmission, and how they differ from other scaffold proteins in this aspect. Here, we investigate the role of Shanks in regulating glutamatergic synaptic transmission at rat hippocampal SC-CA1 synapses, using lentivirus-mediated knockdown and molecular replacement combined with dual whole-cell patch clamp in hippocampal slice culture. In line with previous findings regarding PSD-MAGUK scaffold manipulation, we found that loss of scaffold proteins via knockdown of Shank1 or Shank2, but not Shank3, led to a reduction of the number but not the unitary response of AMPAR-containing synapses. Only when both Shank1 and Shank2 were knocked down, were both the number and the unitary response of active synapses reduced. This reduction was accompanied by a decrease in NMDAR-mediated synaptic response, indicating more profound deficits in synaptic transmission. Molecular replacement with Shank2 and Shank3c rescued the synaptic transmission to the basal level, and the intact sterile α-motif (SAM) of Shank proteins is required for maintaining glutamatergic synaptic transmission. We also found that altered neural activity did not influence the effect of Shank1 or Shank2 knockdown on AMPAR synaptic transmission, in direct contrast to the activity dependence of the effect of PSD-95 knockdown, revealing differential interaction between activity-dependent signaling and scaffold protein families in regulating synaptic AMPAR function.

In B lymphocytes, the uracil N-glycosylase (UNG) excises genomic uracils made by activation-induced deaminase (AID), thus underpinning antibody gene diversification and oncogenic chromosomal translocations, but also initiating faithful DNA repair. Ung-/- mice develop B-cell lymphoma (BCL). However, since UNG has anti- and pro-oncogenic activities, its tumor suppressor relevance is unclear. Moreover, how the constant DNA damage and repair caused by the AID and UNG interplay affects B-cell fitness and thereby the dynamics of cell populations in vivo is unknown. Here, we show that UNG specifically protects the fitness of germinal center B cells, which express AID, and not of any other B-cell subset, coincident with AID-induced telomere damage activating p53-dependent checkpoints. Consistent with AID expression being detrimental in UNG-deficient B cells, Ung-/- mice develop BCL originating from activated B cells but lose AID expression in the established tumor. Accordingly, we find that UNG is rarely lost in human BCL. The fitness preservation activity of UNG contingent to AID expression was confirmed in a B-cell leukemia model. Hence, UNG, typically considered a tumor suppressor, acquires tumor-enabling activity in cancer cell populations that express AID by protecting cell fitness.

What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, by genetic and structural analysis, we demonstrate that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3. This interaction protects LGI1-ADAM22 from endocytosis-dependent degradation. Accordingly, forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we find that ∼50% of LGI1 and ∼10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment.

Human genome-wide association studies have linked single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 near the INK4/ARF (CDKN2a/b) locus with susceptibility to atherosclerotic vascular disease (ASVD). Although this locus encodes three well-characterized tumor suppressors, p16(INK4a), p15(INK4b), and ARF, the SNPs most strongly associated with ASVD are ∼120 kb from the nearest coding gene within a long non-coding RNA (ncRNA) known as ANRIL (CDKN2BAS). While individuals homozygous for the atherosclerotic risk allele show decreased expression of ANRIL and the coding INK4/ARF transcripts, the mechanism by which such distant genetic variants influence INK4/ARF expression is unknown. Here, using rapid amplification of cDNA ends (RACE) and analysis of next-generation RNA sequencing datasets, we determined the structure and abundance of multiple ANRIL species. Each of these species was present at very low copy numbers in primary and cultured cells; however, only the expression of ANRIL isoforms containing exons proximal to the INK4/ARF locus correlated with the ASVD risk alleles. Surprisingly, RACE also identified transcripts containing non-colinear ANRIL exonic sequences, whose expression also correlated with genotype and INK4/ARF expression. These non-polyadenylated RNAs resisted RNAse R digestion and could be PCR amplified using outward-facing primers, suggesting they represent circular RNA structures that could arise from by-products of mRNA splicing. Next-generation DNA sequencing and splice prediction algorithms identified polymorphisms within the ASVD risk interval that may regulate ANRIL splicing and circular ANRIL (cANRIL) production. These results identify novel circular RNA products emanating from the ANRIL locus and suggest causal variants at 9p21.3 regulate INK4/ARF expression and ASVD risk by modulating ANRIL expression and/or structure.