Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

<div><p>Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (<em>Ambystoma mexicanum</em>) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that <em>miR-21</em> was overexpressed in mid-bud blastemas compared to stump tissue. Mature <em>A. mexicanum (“Amex”) miR-21</em> was detected in axolotl RNA by Northern blot and differential expression of <em>Amex-miR-21</em> in blastema versus stump was confirmed by quantitative RT-PCR. We identified the <em>Amex Jagged1</em> as a putative target gene for <em>miR-21</em> during salamander limb regeneration. We cloned the full length 3′UTR of <em>Amex-Jag1</em>, and our <em>in vitro</em> assays demonstrated that its single <em>miR-21</em> target recognition site is functional and essential for the response of the <em>Jagged1</em> gene to <em>miR-21</em> levels. Our findings pave the road for advanced <em>in vivo</em> functional assays aimed to clarify how microRNAs such as <em>miR-21</em>, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as <em>Jagged1</em> during regenerative processes.</p> </div

Fold changes of the most significant differentially-expressed miRNAs in salamander limb regeneration.

<p>Differentially expressed microRNAs that passed the filtering criteria on variation across samples (<i>p</i>≤0.001). Each bar indicates the fold change between blastema (Bl) and stump (St) samples. The y-axis is a log scale. A fold change >1 indicates up-regulation in Bl samples (red bars) and a fold change <1 indicates down-regulation in Bl samples (blue bars) compared to St samples. Three consecutive asterisks indicate miR-21 probes.</p

Jagged1 as a putative target of Amex-miR-21.

<p>A. Comparison between the human and axolotl miR-21 target sites in their Jagged1 genes. Nucleotide alignment of the <i>miR-21</i> target site-containing region present in the 3′-UTR of the human <i>Jagged1</i> (<i>Hsa-Jag1</i>, NM_000214) and the axolotl <i>Jagged1</i> (<i>Amx-Jag1</i>, JF907581). The 22 bases comprising the target site for <i>miR-21</i> are in yellow except for the 7 bases complementary to the <i>miR-21</i> seed region (green color). Vertical bars (|) denote nucleotide identities between the two sequences. The numbers at both sides of the alignment are the nucleotide position on each 3′-UTR. B. <i>In vitro</i> luciferase assays testing the effect of <i>miR-21</i> on the 3′-UTR of <i>Amex-Jag1 in axolotl cells</i>. Results are expressed as percent of co-reporter-normalized <i>Renilla</i> activity against reference vectors. Bars denote standard error of mean of the amount of independent assays. Student <i>t-</i>test was done and the obtained <i>p-</i>values determined that the <i>Target of Hsa-miR-21</i> is a good positive control as biosensor for the activity of this microRNA (<i>p</i>≤0.005; two-tailed <i>t-</i>test). These results suggest that <i>Amex-Jag1</i> may in fact be a target for <i>miR-21</i> because significant differences were found in the <i>Renilla</i> signal recorded from cells electroporated with only the vector containing the 3′-UTR of <i>Amex-Jag1</i>, versus the cells that were also electroporated with Pre-miR-21 *** (p≤0.005) but not Pre-miR30a. When the latter experiment was repeated with the mutant, <i>seedless</i>-<i>Amex-Jag1-</i>3′-UTR, any sensitivity to exogenous <i>miR-21</i> was lost.</p

Identification of MAC1: A Small Molecule That Rescues Spindle Bipolarity in Monastrol-Treated Cells

The genetic integrity of each organism is intimately tied to the correct segregation of its genome during mitosis. Insights into the underlying mechanisms are fundamental for both basic research and the development of novel strategies to treat mitosis-relevant diseases such as cancer. Due to their fast mode of action, small molecules are invaluable tools to dissect mitosis. Yet, there is a great demand for novel antimitotic compounds. We performed a chemical genetic suppression screen to identify compounds that restore spindle bipolarity in cells treated with Monastrol, an inhibitor of the mitotic kinesin Eg5. We identified one compoundMAC1that rescued spindle bipolarity in cells lacking Eg5 activity. Mechanistically, MAC1 induces the formation of additional microtubule nucleation centers, which allows kinesin Kif15-dependent bipolar spindle assembly in the absence of Eg5 activity. Thus, our chemical genetic suppression screen revealed novel unexpected insights into the mechanism of spindle assembly in mammalian cells

Assessing Different E3 Ligases for Small Molecule Induced Protein Ubiquitination and Degradation

Proteolysis targeting chimera (PROTAC) technology, the recruitment of E3 ubiquitin ligases to induce the degradation of a protein target, is rapidly impacting chemical biology, as well as modern drug development. Here, we explore the universality of this approach by evaluating different E3 ubiquitin ligases, engineered in their substrate binding domains to accept a recruiting ligand. Five out of six E3 ligases were found to be amenable to recruitment for target degradation. Taking advantage of the tight spatiotemporal control of inducing ubiquitination on a preselected target in living cells, we focused on two of the engineered E3 ligases, βTRCP and parkin, to unravel their ubiquitination characteristics in comparison with the PROTAC-recruited endogenous E3 ligases VHL and cereblon

A HaloTag-Based Small Molecule Microarray Screening Methodology with Increased Sensitivity and Multiplex Capabilities

Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule–protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD’s “bump” ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck

HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knock down widely used HaloTag7 fusion proteins in a general fashion

Targeting the von Hippel–Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1α Interaction

E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein–protein interactions. Using rational design, we have generated the first small molecule targeting the von Hippel–Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compound mimics the binding mode of the transcription factor HIF-1α, a substrate of VHL. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of chronic anemia and ischemia

Targeting the C481S Ibrutinib-Resistance Mutation in Bruton’s Tyrosine Kinase Using PROTAC-Mediated Degradation

Inhibition of Bruton’s tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide a basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL

Targeting the C481S Ibrutinib-Resistance Mutation in Bruton’s Tyrosine Kinase Using PROTAC-Mediated Degradation

Inhibition of Bruton’s tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide a basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL