Paul Chang

Poly(ADP-Ribose) Regulates Stress Responses and MicroRNA Activity in the Cytoplasm

Poly(ADP-ribose) is a major regulatory macromolecule in the nucleus, where it regulates transcription, chromosome structure, and DNA damage repair. Functions in the interphase cytoplasm are less understood. Here, we identify a requirement for poly(ADP-ribose) in the assembly of cytoplasmic stress granules, which accumulate RNA-binding proteins that regulate the translation and stability of mRNAs upon stress. We show that poly(ADP-ribose), six specific poly(ADP-ribose) polymerases, and two poly(ADP-ribose) glycohydrolase isoforms are stress granule components. A subset of stress granule proteins, including microRNA-binding Argonaute family members Ago1-4, are modified by poly(ADP-ribose), and such modification increases upon stress, a condition when both microRNA-mediated translational repression and microRNA-directed mRNA cleavage are relieved. Similar relief of repression is also observed upon overexpression of specific poly(ADP-ribose) polymerases or, conversely, upon knockdown of glycohydrolase. We conclude that poly(ADP-ribose) is a key regulator of posttranscriptional gene expression in the cytoplasm.

Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function

Poly(ADP-ribose) (PAR) is a large, negatively charged post-translational modification that is produced by polymerization of NAD+ by PAR polymerases (PARPs). There are at least 18 PARPs in the human genome, several of which have functions that are unknown. PAR modifications are dynamic; PAR structure depends on the balance between synthesis and hydrolysis by PAR glycohydrolase. We previously found that PAR is enriched in vertebrate somatic-cell mitotic spindles and demonstrated a requirement for PAR in the assembly of Xenopus egg extract spindles. Here, we knockdown all characterized PARPs using RNA interference (RNAi), and identify tankyrase-1 as the PARP that is required for mitosis. Tankyrase-1 localizes to mitotic spindle poles, to telomeres and to the Golgi apparatus. Tankyrase-1 RNAi was recently shown to result in mitotic arrest, with abnormal chromosome distributions and spindle morphology observed — data that is interpreted as evidence of post-anaphase arrest induced by failure of telomere separation. We show that tankyrase-1 RNAi results in pre-anaphase arrest, with intact sister-chromatid cohesion. We also demonstrate a requirement for tankyrase-1 in the assembly of bipolar spindles, and identify the spindle-pole protein NuMA as a substrate for covalent modification by tankyrase-1.

Poly(ADP-ribose) is required for spindle assembly and structure

The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs). Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function. Here we show that PAR is enriched in the spindle and is required for spindle function—PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.

Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes

Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by ϵ-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, our data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole.

δ-Tubulin and ε-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function

The centrosome organizes microtubules, which are made up of α-tubulin and β-tubulin, and contains centrosome-bound γ-tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that they are associated with the centrosome. One is a homologue of the Chlamydomonas δ-tubulin Uni3, and the other is a new tubulin, which we have named ɛ-tubulin. Localization of δ-tubulin and ɛ-tubulin to the centrosome is independent of microtubules, and the patterns of localization are distinct from each other and from that of γ-tubulin. δ-Tubulin is found in association with the centrioles, whereas ɛ-tubulin localizes to the pericentriolar material. ɛ-Tubulin exhibits a cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated pair and later associating with both centrosomes. ɛ-Tubulin thus distinguishes the old centrosome from the new at the level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the recruitment of ɛ-tubulin.

Epsilon-tubulin is required for centriole duplication and microtubule organization.

Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified ɛ-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of ɛ-tubulin to the new centrosome can only occur after exit from S phase and that ɛ-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis ɛ-tubulin was cloned and shown to be similar to human ɛ-tubulin in both sequence and localization. Depletion of ɛ-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that ɛ-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that ɛ-tubulin is required for centriole duplication and organization of the pericentriolar material.

Use and Associated Risks of Concomitant Aspirin Therapy With Oral Anticoagulation in Patients With Atrial Fibrillation Insights From the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF) Registry

Atrial fibrillation (AF) represents the most common arrhythmia in the US, and it substantially increases the risk of stroke.1, 2 Oral anticoagulant (OAC) therapy is the mainstay of treatment for AF patients at risk for stroke. Many patients with AF also have coexistent atherosclerotic cardiovascular disease,3 and may be also put on antiplatelet therapy in addition to OAC medications. However, the incremental benefit of antiplatelet therapy added to anticoagulation in patients with AF is unclear. While European guidelines support the use of more aggressive concomitant antiplatelet therapy over short periods of time in patients at acceptably low risk for bleeding, US guidelines are more reserved.4, 5 To date, there have been limited data available to define current patterns of use of concomitant antiplatelet therapy along with OAC in AF patients in the US. Furthermore, the risks of such combinations in community practice remain poorly defined. To address these important questions, we used data from the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF) to investigate contemporary implementation of aspirin therapy in patients with AF receiving OAC, and associated clinical outcomes. Our objectives were to:(1) describe overall use of concomitant antiplatelet and OAC therapy in AF patients; (2) identify clinical factors associated with concomitant therapy; (3) note use of dual therapy among those without any known cardiovascular disease; and (4) determine if the addition of antiplatelet therapy is associated with risk for subsequent bleeding events.Background— The role of concomitant aspirin (ASA) therapy in patients with atrial fibrillation (AF) receiving oral anticoagulation (OAC) is unclear. We assessed concomitant ASA use and its association with clinical outcomes among AF patients treated with OAC. Methods and Results— The Outcomes Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF) registry enrolled 10 126 AF patients from 176 US practices from June 2010 through August 2011. The study population was limited to those on OAC (n=7347). Hierarchical multivariable logistic regression models were used to assess factors associated with concomitant ASA therapy. Primary outcomes were 6-month bleeding, hospitalization, ischemic events, and mortality. Overall, 35% of AF patients (n=2543) on OAC also received ASA (OAC+ASA). Patients receiving OAC+ASA were more likely to be male (66% versus 53%; P<0.0001) and had more comorbid illness than those on OAC alone. More than one third of patients (39%) receiving OAC+ASA did not have a history of atherosclerotic disease, yet 17% had elevated Anticoagulation and Risk Factors in Atrial Fibrillation (ATRIA) bleeding risk scores (≥5). Major bleeding (adjusted hazard ratio, 1.53; 95% confidence interval, 1.20–1.96) and bleeding hospitalizations (adjusted hazard ratio, 1.52; 95% confidence interval, 1.17–1.97) were significantly higher in those on OAC+ASA compared with those on OAC alone. Rates of ischemic events were low. Conclusions— Patients with AF receiving OAC are often treated with concomitant ASA, even when they do not have cardiovascular disease. Use of OAC+ASA was associated with significantly increased risk for bleeding, emphasizing the need to carefully determine if and when the benefits of concomitant ASA outweigh the risks in AF patients already on OAC. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01165710.

New PARP targets for cancer therapy

Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD+ as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5A, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, most of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer-relevant functions for these PARPs, which indicates that we need to understand more about these PARPs to effectively target them.

Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

Cardiac gene transfer by intracoronary infusion of adenovirus vector—mediated reporter gene in the transplanted mouse heart

This study introduces a model for intracoronary gene transfer in murine cardiac isografts using adenovirus vectors. This approach may offer an opportunity to modulate alloreactivity after cardiac transplantation. Donor hearts were infected via the coronary arteries with a volume of 10(9) plaque-forming units per milliliter of a recombinant adenovirus containing the beta-galactosidase-encoding gene (Ad.CMVLacZ). In a control group, 200 microliters of normal saline solution was infused. The grafts were stored in 4 degrees C cold saline solution for 15 minutes, then transplanted heterotopically into syngeneic hosts (B10.BR). The grafts were harvested at 3, 7, 15, or 30 days (n = 5 for each group) after transplantation, and beta-galactosidase activity was assessed by histochemical staining (X-gal). All grafts were functioning when harvested. X-gal staining pattern was nonuniform with positive staining appearing in epicardial, myocardial, and endocardial cells, as well as in the vessel walls. The cells permissive to infection consisted predominantly of myocardial cells. The mean total numbers of beta-gal-positive staining cells per slice were 68.7 +/- 27.3 in the 3-day group, 330.4 +/- 53.8 in the 7-day group, 151.3 +/- 48.0 in the 15-day group, and 39.9 +/- 10.8 in the 30-day group, thus peaking in the 7-day group (p < 0.05). Control isografts (n = 5), retrieved at day 30, revealed no staining activity. In conclusion, our model demonstrates that intracoronary gene transfer to the transplanted murine cardiac grafts is feasible at the time of harvest. Adenovirus-mediated gene transfer produces widespread gene expression which, though perhaps transient, does not adversely affect myocardial structure or function. This technology may allow modification of graft immunogenicity in the future through the production of therapeutic proteins sufficient to modulate local immune responses.