Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

Rho regulation: DLC proteins in space and time.

Rho GTPases function as molecular switches that connect changes of the external environment to intracellular signaling pathways. They are active at various subcellular sites and require fast and tight regulation to fulfill their role as transducers of extracellular stimuli. New imaging technologies visualizing the active states of Rho proteins in living cells elucidated the necessity of precise spatiotemporal activation of the GTPases. The local regulation of Rho proteins is coordinated by the interaction with different guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that turn on and off GTPase signaling to downstream effectors. GEFs and GAPs thus serve as critical signaling nodes that specify the amplitude and duration of a particular Rho signaling pathway. Despite their importance in Rho regulation, the molecular aspects underlying the spatiotemporal control of the regulators themselves are still largely elusive. In this review we will focus on the Deleted in Liver Cancer (DLC) family of RhoGAP proteins and summarize the evidence gathered over the past years revealing their different subcellular localizations that might account for isoform-specific functions. We will also highlight the importance of their tightly controlled expression in the context of neoplastic transformation.

The Rho-specific GAP protein DLC3 coordinates endocytic membrane trafficking.

Membrane trafficking is known to be coordinated by small GTPases, but the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure balanced GTPase activation at different subcellular sites is largely elusive. Here, we show in living cells that deleted in liver cancer 3 (DLC3, also known as STARD8) is a functional Rho-specific GAP protein, the loss of which enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and is required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment (ERC), which is restored by the simultaneous downregulation of RhoA and RhoB. We further demonstrate that DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation associated with prolonged receptor signaling. Taken together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport through the control of Rho activity.

Directed HIV-1 evolution of protease inhibitor resistance by second-generation short hairpin RNAs.

Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches.

Health service utilization in patients with major depression and co-morbid pain.

AIMS: Patients with depression often have co-morbid pain symptoms. However, rates of service utilization by psychiatric in-patients with co-morbid pain symptoms are unknown. The purpose of this study is to estimate whether patients with major depression and co-morbid pain access medical treatment for their pain as much as their counterparts with psychiatric diagnoses other than major depression. METHODS: A total of 103 patients (62 female; 41 male) were assessed for a diagnosis of major depression applying a psychiatric clinical interview followed by a self-report pain questionnaire, which assessed physical pain in psychiatric patients. RESULTS: Patients with major depression reported higher rates of pain symptoms in the past 6 and 12 months than their counterparts with a psychiatric diagnosis other than major depression. Analysis of variance showed that patients with depression were less likely to attend medical and specialist services for their pain symptoms than their counterparts. On the contrary, depressed patients with pain attended more frequently general in-patient services than non-depressed patients with pain. CONCLUSIONS: Patients with depression suffer high rates of pain symptoms, but are at higher risk of not accessing appropriate services suggesting inadequate service utilization. The results have implications for screening and health care delivery for psychiatric patients with pain.

Cognitive dysfunction in mice deficient for TNF- and its receptors.

Recent evidence suggests a role for tumor necrosis factor alpha (TNF) in the functioning of the central nervous system (CNS). The aim of this work was to examine the effect of a deficiency of TNF (TNF(-/-)) and its main receptors (TNF-R1(-/-) and TNF-R2(-/-)) on cognitive function. A standardized survey on cognition-like behavior assessing learning and retention, spatial learning/memory, cognitive flexibility, and learning effectiveness was used in B6.WT and B6.TNF gene targeted mice strains (B6.wild-type, B6.TNF(-/-), B6.TNF-R1(-/-), B6.TNF-R2(-/-) mice). All studied mice strains demonstrated successful exploration and learning processes during the training phases of the tests, which made the specific cognition-like tests valid in these mice strains. In the specific cognition-like tests, the B6.TNF(-/-) mice demonstrated significantly poorer learning and retention in the novel object test compared to B6.WT, B6.TNF-R1(-/-) and B6.TNF-R2(-/-) mice. In addition, spatial learning and learning effectiveness were significantly poorer in B6.TNF(-/-) mice compared to B6.WT mice. Moreover, the moderately impaired cognitive performance with similar degrees in B6.TNF-R1(-/-) or B6.TNF-R2(-/-) mice was generally better than in TNF(-/-) mice but also poorer than in B6.WT mice. While the absence of TNF was correlated with poor cognitive functioning, the deletion of both TNF-receptors was involved in partially reduced cognitive functioning. Low-levels of TNF under non-inflammatory immune conditions appear essential for normal cognitive function. TNF displays an interesting candidate gene for cognitive function. Translational research is required to investigate associations between genetic variants of TNF and cognitive function in healthy subjects and neuropsychiatric samples.

Germline mutations of the MSR1 gene in prostate cancer families from Germany.

The MSR1 gene at 8p22 has been suggested as a candidate gene for hereditary prostate cancer because germline variants have been found to be associated with the disease. Aside from a single nonsense mutation (R293X) that was found repeatedly at low frequencies in several samples, little evidence has been gained by follow-up studies to confirm the gene's relevance for prostate cancer. Prompted by reasonable support for a linkage to 8p22, we sought to determine the mutation spectrum of MSR1 in our family sample. Screening of 139 probands (representing 139 prostate cancer families) revealed 15 novel and a total of 20 sequence variants within the 10 coding exons and their intronic proximities. Aside from the known mutation c.877C>T (R293X) present in two of our families, we identified a second nonsense allele (c.251C>G; S84X) and a splice-site mutation (c.818-1G>A) that results in mRNA instability (each in a single pedigree). The novel missense alleles were c.703C>T (H235Y), c.856C>T (P286S), c.905C>T (P302L), c.1193C>G (A398G), and c.1289A>G (K430R). Of the eight variants that affect the encoded protein (splice site, nonsense, and missense), only R293X as well as the polymorphism c.823C>G (P275A) were additionally present at remarkable frequencies in further samples of sporadic prostate cancer and controls. Of note, carriers of R293X were equally frequent in 367 sporadic prostate cancer cases (1.9%) and in 197 controls (2.0%). To our knowledge, our study is the first to demonstrate further loss of function variants of MSR1 apart from R293X. Nevertheless, the low frequencies of deleterious alleles, in addition to an apparently moderate penetrance, does not support MSR1 as a major susceptibility gene in this family sample.