Structural basis for p50RhoGAP BCH domain–mediated regulation of Rho inactivation

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the β5-strand of the BCH domain is involved in an intermolecular β-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the β5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain–containing proteins.

Small GTPases are molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state and are primarily involved in cytoskeletal reorganization during cell motility, morphogenesis, and cytokinesis (1, 2). These small GTPases are tightly controlled by activators and inactivators, such as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively (3, 4), which are multidomain proteins that are themselves regulated through their interactions with other proteins, lipids, secondary messengers, and/or by posttranslational modifications (5–7). Despite our understanding of the mechanisms of action of GTPases, GAPs, and GEFs, little is known about how they are further regulated by other cellular proteins in tightly controlled local environments.The BNIP-2 and Cdc42GAP Homology (BCH) domain has emerged as a highly conserved and versatile scaffold protein domain that targets small GTPases, their GEFs, and GAPs to carry out various cellular processes in a spatial, temporal, and kinetic manner (8–15). BCH domain–containing proteins are classified into a distinct functional subclass of the CRAL_TRIO/Sec14 superfamily, with ∼175 BCH domain–containing proteins (in which 14 of them are in human) present across a range of eukaryotic species (16). Some well-studied BCH domain–containing proteins include BNIP-2, BNIP-H (CAYTAXIN), BNIP-XL, BNIP-Sα, p50RhoGAP (ARHGAP1), and BPGAP1 (ARHGAP8), with evidence to show their involvement in cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, growth activation and suppression, myoblast differentiation, and neuritogenesis (17–21). Aside from interacting with small GTPases and their regulators, some of these proteins can also associate with other signaling proteins, such as fibroblast growth factor receptor tyrosine kinases, myogenic Cdo receptor, p38-MAP kinase, Mek2/MP1, and metabolic enzymes, such as glutaminase and ATP-citrate lyase (17–26). Despite the functional diversity and versatility of BCH domain–containing proteins, the structure of the BCH domain and its various modes of interaction remain unknown. The BCH domain resembles the Sec14 domain (from the CRAL-TRIO family) (16, 27, 28), a domain with lipid-binding characteristics, which may suggest that the BCH domain could have a similar binding strategy. However, to date, the binding and the role of lipids in BCH domain function remain inconclusive.Of the BCH domain–containing proteins, we have focused on the structure and function of p50RhoGAP. p50RhoGAP comprises an N-terminal BCH domain and a C-terminal GAP domain separated by a proline-rich region. We found that p50RhoGAP contains a noncanonical RhoA-binding motif in its BCH domain and is associated with GAP-mediated cell rounding (13). Further, we showed previously that deletion of the BCH domain dramatically enhanced the activity of the adjacent GAP domain (13); however, the full dynamics of this interaction is unclear. Previously, it has been reported that the BCH and other domains regulate GAP activity in an autoinhibited manner (18, 21, 29, 30) involving the interactions of both the BCH and GAP domains, albeit the mechanism remains to be investigated. It has also been shown that a lipid moiety on Rac1 (a Rho GTPase) is necessary for its inactivation by p50RhoGAP (29, 31), which may imply a role in lipid binding. An understanding of how the BCH domain coordinates with the GAP domain to affect the local activity of RhoA and other GTPases would offer a previously unknown insight into the multifaceted regulation of Rho GTPase inactivation.To understand the BCH domain–mediated regulation of p50RhoGAP and RhoA activities, we have determined the crystal structure of a homologous p50RhoGAP BCH domain from S. pombe for functional interrogation. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-interacting loop and a lipid-binding pocket. Our results show that the lipid-binding region of the BCH domain helps to anchor the prenylation tail of RhoA while the loop interacts directly with RhoA. Moreover, we show that a mutation in the β5-strand releases the autoinhibition of the GAP domain by the BCH domain. This renders the GAP domain active, leading to RhoA inactivation and the associated phenotypic effects in yeast and HeLa cells. The released BCH domain also contributes to enhanced p50RhoGAP–p50RhoGAP interaction. Our findings offer crucial insights into the regulation of Rho signaling by BCH domain–containing proteins.     

Transverse stiffness: a method for estimation of myocardial wall stress

Determination of regional ventricular wall stress would allow quantification of both regional contractile state and its interplay with global function. Current methods for quantifying regional stress include mathematical modelling and measurements with strain gauges. Both methods are difficult to validate. We hypothesized that transverse stiffness (i.e., the ratio of indentation stress to strain as the ventricular wall is indented in the direction perpendicular to the wall) would be proportional to the stresses in the plane of the wall and could be used to estimate the latter. To test this hypothesis, 6 arterially perfused canine ventricular septa were mounted in an apparatus that could exert biaxial load in the plane of the wall. A servo system maintained the central third of the septa isometric during active contractions while the septa were paced at 30-60 pulses/min. In the center of the isometric region, a probe of 7 mm diameter indented the septa while the transverse indentation stress and strain were measured. For values of peak systolic in-plane stress from 0.56 to 2.6 g/mm2, the transverse stiffness varied from 1.2 to 11.7 g/mm2 and was linearly related to the in-plane wall stress in each septum (p less than 0.001, ANOVA). After cardioplegia, the transverse stiffness also correlated with passively applied wall stress for each dog (p less than 0.001). The slopes of the individual relations between transverse stiffness and wall stress from active contractions were similar to those from passively applied stress (mean +/- SEM; 1.82 +/- 0.36 versus 1.45 +/- 0.31, NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and specific immunization against TNFalpha confers no additional benefit

TNFalpha participates in the pathogenesis of atherosclerosis. The effect of immunization against TNFalpha on development of advanced vascular lesions in atherosclerosis-susceptible apoE-deficient mice was investigated. At 5-7 weeks of age, animals received immunization with either Freunds adjuvant and a recombinant antigenic TNFalpha molecule (TNF106), Freunds adjuvant alone, or no immunization. All mice received a Western-type high-fat diet for 12 weeks. Aortic sinus lesion area was determined by microscopic morphometry, the total aortic arch cholesterol content was determined by gas chromatography, and antibodies against TNFalpha, malondialdehyde-modified low density lipoprotein, or heat shock protein 60, were assessed by ELISAs. Immunization with TNF106 induced high-titered circulating antibodies against TNFalpha (n=23), and these antibodies were not detected in mice immunized with Freunds adjuvant alone (n=22), or in non-immunized control animals (n=25). After 12 weeks, the atherosclerotic lesion size was significantly reduced in immunized animals, whether they had been immunized with TNF106 or Freunds adjuvant alone, and the total lesional cholesterol content was decreased in mice immunized with TNF106. There were no correlations between circulating antibody titers and plaque size, total aortic arch cholesterol content, or plasma lipid levels, respectively. Administration of Freunds adjuvant alone can thus reduce formation of mature atherosclerotic lesions in apoE-deficient mice and this response is not modified by specific immunization against TNFalpha.     

Comparison of biodegradable and newer generation durable polymer drug-eluting stents with short-term dual antiplatelet therapy: a systematic review and Bayesian network meta-analysis of randomized trials comprising of 43,875 patients

Journal of Thrombosis and Thrombolysis - Newer generation durable polymer drug-eluting stents (DP-DES) and biodegradable polymer DES (BP-DES) have similar efficacy with dual-antiplatelet therapy...     

Using a telephone support group for HIV-positive persons aged 50+ to increase social support and health-related knowledge

Middle-aged and older persons living with HIV/AIDS have unique needs arising from the physical, mental, and social changes associated not only with normal aging but also related to living with a chronic illness. To address these needs, two 10-week telephone psychoeducational support groups were offered for HIV-infected persons aged 50 or older. Each group was cofacilitated by a registered nurse and a social worker; each session was 50-60 minutes every Friday; approximately 1-5 clients participated with an average number of 3 clients and there was no charge to the participants. The issues addressed in the group were: (1) staying healthy; (2) symptom management; (3) understanding other chronic illnesses; (4) understanding diagnostic tests; (5) strategies for effective interactions with the health care provider; (6) optimizing HIV/AIDS medication use; (7) understanding new developments in HIV treatment; (8) coping with losses; and (9) finding commonalities. There were unique challenges. Boundaries of respect were more difficult to maintain in a teleconference, as opposed to an in-person group. Nonverbal cues were impossible to interpret and therefore greater sensitivity was required to gauge the impact of borderline, less controlled group members, especially in relationship to other group members who may tend to be less assertive. One group member withdrew because his hearing was impaired and the telephone modality was just too challenging. It has been found that middle-aged and older adults living with HIV/AIDS with greater depression identify a need for information and support. It is crucial to share concrete information, identify symptoms clearly, and explore the use of effective and ineffective medications and treatments. The psychosocial concerns are very real and encouraging group members to open up to one another to create a cohesive community of sharing is equally important. Although the use of teleconference technology makes this more difficult, the attempt to create a situation that facilitates connections with another individual is one strategy to decrease geographic or logistical isolation.