On the asymmetric partitioning of nucleocytoplasmic transport - recent insights and open questions.

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the ß-karyopherin (Kapß) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP-RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapßs, cargoes and Ran to establish the asymmetry of NCT.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

The role of nucleocytoplasmic transport in mechanotransduction.

Cells integrate mechanical and biochemical signals via a process called mechanotransduction to generate essential gene expression patterns in space and time. This is vital for cell migration and proliferation as well as tissue morphogenesis and remodeling. While the force-sensing and force-transducing mechanisms are generally known, it remains unclear how mechanoresponsive transcription factors (TFs) are selectively translocated into the nucleus upon force activation. Such TFs include Yes-Associated Protein (YAP), Myocardin Related Transcription Factors (MRTFs), Hypoxia Induced Factors (HIFs) and others. Here, we discuss how the nucleocytoplasmic transport machinery intersects with mechanoresponsive TFs to facilitate their selective transport through nuclear pore complexes.

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera.

Fungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based on cytochrome c oxidase subunit 1 and ß-tubulin genes. The assays resulted in 2-10 restriction patterns, depending on the gene amplified and restriction enzyme applied. Pooled analysis of the patterns allowed to propose an algorithm, that can be successfully used for an accurate species-specific identification of 21 species of the Scopulariopsis-like fungi.

Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains.

Resistance of Mycobacterium tuberculosis to rifampin (RMP), mediated by mutations in the rpoB gene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individual rpoB mutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout the rpoB gene among 115 Mycobacterium tuberculosis clinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discovered rpoB mutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results from in vitro mutagenesis experiments were combined with the assessment of the prevalence of rpoB mutations, and their reciprocal co-occurrences, across global M. tuberculosis populations. Twenty-two different types of mutations in the rpoB gene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50 and MIC90 values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenic M. tuberculosis H37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

High chymotrypsin-like activity in the plasma of patients with newly diagnosed multiple myeloma treated with bortezomib is predictive of a better response and longer PFS.

Proteasome inhibitors (PIs) such as bortezomib constitute an important part of the modern standard therapy for multiple myeloma (MM). In this study, we set out to assess whether proteasome concentration and chymotrypsin-like (ChT-L) activity could serve as potential biomarkers defining the likelihood of response to treatment with bortezomib, in order to identify patients who are more likely to respond to treatment with PI. We analysed proteasome concentration and ChT-L activity in the plasma of 78 patients with newly diagnosed MM during treatment with or without proteasome inhibitors. Values of all the studied parameters in the group of responders decreased sharply from the initial levels already after the third cycle of chemotherapy and remained significantly lower until the end of treatment. On the other hand, in the group of non-responders, there was an increase in the measured proteasome parameters already after the third cycle, and they remained high during the next cycles of therapy. We also showed that high baseline proteasome ChT-L activity values might prognosticate longer progression-free survival (PFS) in patients treated with PI. Our findings demonstrate that measuring plasma proteasome ChT-L activity can be used as a powerful biomarker for predicting clinical response to treatment and PFS in patients with newly diagnosed MM.

Proteasome 20S in multiple myeloma: comparison of concentration and chymotrypsin-like activity in plasma and serum.

The ubiquitin-proteasome system is relevant in the pathobiology of many haematological malignancies, including multiple myeloma. The assessment of proteasome concentration and chymotrypsin-like (ChT-L) activity might constitute a new approach to diagnosis, prognosis and monitoring of anticancer treatment of patients with haematological malignancies and other diseases. The aim of our study was to determine which material, plasma or serum, is better for measuring chymotrypsin-like (ChT-L) activity and proteasome concentration. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in 70 plasma and serum samples drawn from 28 patients at different treatment stages for multiple myeloma (MM) and 31 healthy volunteers. Proteasome ChT-L activity and concentration in multiple myeloma patients were significantly higher in plasma compared to serum. In this group we observed significant and positive correlations both between the plasma and serum proteasome ChT-L activity and plasma and serum proteasome concentration. The higher values of proteasome concentration and ChT-L activity in plasma than in serum and their better correlations with parameters of tumour load and prognosis suggest that plasma constitutes a better biological material for measuring ChT-L activity and proteasome concentration than serum in multiple myeloma patients.

Mechanism of exportin retention in the cell nucleus.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinß1 (Kapß1 or importinß1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapß1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Karyopherin enrichment and compensation fortifies the nuclear pore complex against nucleocytoplasmic leakage.

Nuclear pore complexes (NPCs) discriminate nonspecific macromolecules from importin and exportin receptors, collectively termed "karyopherins" (Kaps), that mediate nucleocytoplasmic transport. This selective barrier function is attributed to the behavior of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups) that guard the NPC channel. However, NPCs in vivo are typically enriched with different Kaps, and how they impact the NPC barrier remains unknown. Here, we show that two major Kaps, importinß1/karyopherinß1 (Kapß1) and exportin 1/chromosomal maintenance 1 (CRM1), are required to fortify NPC barrier function in vivo. Their enrichment at the NPC is sustained by promiscuous binding interactions with the FG Nups, which enable CRM1 to compensate for the loss of Kapß1 as a means to maintain NPC barrier function. However, such a compensatory mechanism is constrained by the cellular abundances and different binding kinetics for each respective Kap, as evidenced for importin-5. Consequently, we find that NPC malfunction and nucleocytoplasmic leakage result from poor Kap enrichment.

Molecular taxonomy of scopulariopsis-like fungi with description of new clinical and environmental species.

The taxonomy of scopulariopsis-like fungi, comprising numerous human opportunistic species, has recently been reassessed with delineation of the genera Microascus, Pithoascus, Pseudoscopulariopsis, and Scopulariopsis, using morphological data and multilocus sequence analysis based on four loci (ITS, LSU, EF-1α, and TUB). In this study, the same genetic markers were used to investigate a set of clinical and environmental isolates, morphologically identified as Microascus and Scopulariopsis spp. The ingroups of the concatenated phylogenetic tree resolved 41 species clades, with isolates distributed in four main lineages corresponding to the genera Microascus, Pithoascus, Scopulariopsis, and newly established genus Fuscoannellis, typified by Scopulariopsis carbonaria. The new species Microascus chinensis, Microascus onychoides, Microascus pseudolongirostris, Pithoascus lunatus, and Scopulariopsis macurae were described. Microascus trigonosporus var. terreus and Scopulariopsis alboflavescens were found different from M. trigonosporus and Scopulariopsis brevicaulis, respectively. All the species identified in the study, except Fuscoannellis carbonaria and S. macurae, originated from clinical samples, suggesting their potential role in human disease. The use of a four marker combination was demonstrated an efficient and reliable approach to infer phylogenetic relationships among the scopulariopsis-like fungi. Yet, the only genetic marker able to discriminate all species was EF-1α, therefore proposed as a secondary barcode for the identification of these fungi.

Assessment of proteasome concentration and chymotrypsin-like activity in plasma of patients with newly diagnosed multiple myeloma.

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many haematologic malignancies, including multiple myeloma. Under conditions of rapid cell turnover and growth rate, proteasomes are returned into circulation. The measurement of their levels or activity could offer a new approach to diagnosis, prognosis and monitoring of anticancer treatment in carcinoma patients. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in the plasma of 64 patients with a newly diagnosed multiple myeloma and 30 healthy volunteers. The values were found to be significantly higher in the studied patients and advanced disease stages compared to the control group, and decreased significant after chemotherapy. Both proteasome concentration and ChT-L activity correlated with adverse prognostic factors, such as lactate dehydrogenase and ß2-macroglobulin. We also showed that proteasome concentration positively correlates with IL-6 level, as opposed to proteasome ChT-L activity. Of note, higher proteasome ChT-L activity, unlike the concentration, was proved to be an indicator of a shorter progression free survival, constituting thereby an important prognostic marker.