Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer.

Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4-6. Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material-these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers.

WBP11 is required for splicing the TUBGCP6 pre-mRNA to promote centriole duplication.

Centriole duplication occurs once in each cell cycle to maintain centrosome number. A previous genome-wide screen revealed that depletion of 14 RNA splicing factors leads to a specific defect in centriole duplication, but the cause of this deficit remains unknown. Here, we identified an additional pre-mRNA splicing factor, WBP11, as a novel protein required for centriole duplication. Loss of WBP11 results in the retention of ∼200 introns, including multiple introns in TUBGCP6, a central component of the γ-TuRC. WBP11 depletion causes centriole duplication defects, in part by causing a rapid decline in the level of TUBGCP6. Several additional splicing factors that are required for centriole duplication interact with WBP11 and are required for TUBGCP6 expression. These findings provide insight into how the loss of a subset of splicing factors leads to a failure of centriole duplication. This may have clinical implications because mutations in some spliceosome proteins cause microcephaly and/or growth retardation, phenotypes that are strongly linked to centriole defects.

Identification of the Functional Roles of Six Key Proteins in the Biosynthesis of Enterobacteriaceae Colanic Acid.

When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochemically characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liquid chromatography analysis and a fluorescent analogue of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were associated with varying sugar additions or modifications, we developed a liquid chromatography-mass spectrometry method for analysis of the products produced by each enzyme. We have identified the function of all but one encoded glycosyltransferase and have identified the function of two acetyltransferases. This work demonstrates the centrality of acetylation in the biosynthesis of colanic acid and provides insight into the activity of key proteins involved in the production of an important and highly conserved bacterial glycopolymer.

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis.

Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors.

Tuning the production of variable length, fluorescent polyisoprenoids using surfactant-controlled enzymatic synthesis.

Bactoprenyl diphosphate (BPP), a two-E eight-Z configuration C55 isoprenoid, serves as a critical anchor for the biosynthesis of complex glycans central to bacterial survival and pathogenesis. BPP is formed by the polymerase undecaprenyl pyrophosphate synthase (UppS), which catalyzes the elongation of a single farnesyl diphosphate (FPP) with eight Z-configuration isoprene units from eight isopentenyl diphosphates. In vitro analysis of UppS and other polyprenyl diphosphate synthases requires the addition of a surfactant such as Triton X-100 to stimulate the release of the hydrophobic product from the enzyme for effective and efficient turnover. Here using a fluorescent 2-nitrileanilinogeranyl diphosphate analogue of FPP, we have found that a wide range of surfactants can stimulate release of product from UppS and that the structure of the surfactant has a major impact on the lengths of products produced by the protein. Of particular importance, shorter chain surfactants promote the release of isoprenoids with four to six Z-configuration isoprene additions, while larger chain surfactants promote the formation of natural isoprenoid lengths (8Z) and larger. We have found that the product chain lengths can be readily controlled and coarsely tuned by adjusting surfactant identity, concentration, and reaction time. We have also found that binary mixtures of just two surfactants can be used to fine-tune isoprenoid lengths. The surfactant effects discovered do not appear to be significantly altered with an alternative isoprenoid substrate. However, the surfactant effects do appear to be dependent on differences in UppS between bacterial species. This work provides new insights into surfactant effects in enzymology and highlights how these effects can be leveraged for the chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis probes. This work also provides key reagents for the systematic analysis of structure-activity relationships between glycan biosynthesis enzymes and isoprenoid structure.

The metabolic, hormonal, biochemical, and neuromuscular function responses to a backward sled drag training session.

We examined the metabolic, hormonal, biochemical, and neuromuscular function (NMF) responses to a backward sled drag training session (STS) in strength-trained men (n = 11). After baseline collection of saliva (testosterone and cortisol), whole blood (lactate and creatine kinase [CK]), and countermovement jumps (peak power output), participants completed 5 sets of 2 × 20-m (30 second-recovery between drags and 120 second-recovery between sets) maximal backward sled drags (loaded with 75% body mass). Participants were retested immediately, 15 minutes, 1, 3, and 24 hours after STS. Peak power output decreased after STS (baseline, 4,445 ± 705 vs. 0 minute, 3,464 ± 819 W; p = 0.001) and remained below baseline until recovering at both the 3- and 24-hour time points. No changes in CK levels were seen at any time point after STS. Blood lactate increased immediately after STS (baseline, 1.7 ± 0.5 vs. 0 minute, 12.4 ± 2.6 mmol·L-1; p = 0.001) and remained elevated at 60 minutes (3.8 ± 1.9 mmol·L-1; p = 0.004) before returning to baseline at 3 and 24 hours. Testosterone peaked at 15 minutes post (baseline, 158 ± 45 vs. 15 minutes, 217 ± 49 pg·ml-1; p < 0.001) before decreasing below baseline at the 3-hour time point (119 ± 34 pg·ml-1; p = 0.008), but then increased again above baseline at 24 hours (187 ± 56 pg·ml-1; p = 0.04). Cortisol tended to increase at 15 minutes (baseline, 3.4 ± 1.8 vs. 15 minutes, 5.2 ± 2.7 ng·ml-1; p = 0.07) before declining below baseline at 3 hours (1.64 ± 0.93 ng·ml-1; p = 0.012) and returning to baseline concentrations at 24 hours. In conclusion, sled dragging provides an effective metabolic stimulus, with NMF restored after ≤3 hours of recovery. Characterizing the recovery time course after sled training may aid in athlete training program design.