The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production.     

Single or in Combination Antimicrobial Resistance Mechanisms of Klebsiella pneumoniae Contribute to Varied Susceptibility to Different Carbapenems

Resistance to carbapenems has been documented by the production of carbapenemase or the loss of porins combined with extended-spectrum β-lactamases or AmpC β-lactamases. However, no complete comparisons have been made regarding the contributions of each resistance mechanism towards carbapenem resistance. In this study, we genetically engineered mutants of Klebsiella pneumoniae with individual and combined resistance mechanisms, and then compared each resistance mechanism in response to ertapenem, imipenem, meropenem, doripenem and other antibiotics. Among the four studied carbapenems, ertapenem was the least active against the loss of porins, cephalosporinases and carbapenemases. In addition to the production of KPC-2 or NDM-1 alone, resistance to all four carbapenems could also be conferred by the loss of two major porins, OmpK35 and OmpK36, combined with CTX-M-15 or DHA-1 with its regulator AmpR. Because the loss of OmpK35/36 alone or the loss of a single porin combined with bla _CTX-M-15 or bla _DHA-1-ampR expression was only sufficient for ertapenem resistance, our results suggest that carbapenems other than ertapenem should still be effective against these strains and laboratory testing for non-susceptibility to other carbapenems should improve the accurate identification of these isolates.     

Large-scale plant light-use efficiency inferred from the seasonal cycle of atmospheric CO2

We combined atmospheric CO₂ measurements, satellite observations, and an atmospheric transport model in an inverse modeling framework to infer a key property of vegetation physiology, the light-use efficiency (LUE) of net primary production, for large geographic regions. We find the highest LUE in boreal regions and in the northern hemisphere tropics. Within boreal zones, Eurasian LUE is higher than North American LUE and has a distinctly different seasonal profile. This longitudinal asymmetry is consistent with ecological differences expected from the much greater cover of deciduous vegetation in boreal Eurasia caused by the vast Siberian forests of the deciduous conifer, Larch. Inferred LUE of the northern hemisphere tropics is also high and displays a seasonal profile consistent with variations of both cloud cover and C₄ vegetation activity.     

Solution structural studies on human erythrocyte alpha-spectrin tetramerization site

We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid alpha-spectrin. The first 20 residues preceding the first helix (helix C') are in a disordered conformation. The subsequent three helices (helices A1, B1, and C1) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C', the partial domain involved in alpha- and beta-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C' is connected to helix A1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of alpha- and beta-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying alpha- and beta-subunit self-association affinities in their formation of the various spectrin tetramers.     

Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

Coxsackievirus infection induces an abnormal accumulation of ubiquitin aggregates that are generally believed to be noxious to the cells and have a key role in viral pathogenesis. Selective autophagy mediated by autophagy adaptor proteins, including sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 gene 1 protein (NBR1), are an important pathway for disposing of misfolded/ubiquitin conjugates. We have recently demonstrated that SQSTM1 is cleaved after coxsackievirus infection, resulting in the disruption of SQSTM1 function in selective autophagy. NBR1 is a functional homolog of SQSTM1. In this study, we propose to test whether NBR1 can compensate for the compromise of SQSTM1 after viral infection. Of interest, we found that NBR1 was also cleaved after coxsackievirus infection. This cleavage took place at two sites mediated by virus-encoded protease 2A^pro and 3C^pro, respectively. In addition to the loss-of-function, we further investigated whether cleavage of SQSTM1/NBR1 leads to the generation of toxic gain-of-function mutants. We showed that the C-terminal fragments of SQSTM1 and NBR1 exhibited a dominant-negative effect against native SQSTM1/NBR1, probably by competing for LC3 and ubiquitin chain binding. Finally, we demonstrated a positive, mutual regulatory relationship between SQSTM1 and NBR1 during viral infection. We showed that knockdown of SQSTM1 resulted in reduced expression of NBR1, whereas overexpression of SQSTM1 led to increased level of NBR1, and vice versa, further excluding the possible compensation of NBR1 for the loss of SQSTM1. Taken together, the findings in this study suggest a novel mechanism through which coxsackievirus infection induces increased accumulation of ubiquitin conjugates and subsequent viral damage.Autophagy is a conserved biological process and has long been regarded as a non-selective, bulk degradative pathway to maintain the homeostasis of cellular environment under stress and starvation.¹ However, accumulating evidence indicates that autophagy-mediated degradation is more selective than originally thought. Several autophagy adapter proteins, including sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 gene 1 protein (NBR1), have been identified and found to be essential in mediating selective autophagy.^{2, 3} They share a similar domain architecture containing an N-terminal Phox/Bem1p (PB1) domain, a microtubule-associated protein light chain (LC3)-interacting region (LIR), and a C-terminal ubiquitin association (UBA) domain, which allow them to recognize and specifically target substrates to autophagosome for degradation. Although the mechanisms underlying specific substrate selection remain not completely understood, increasing evidence supports that ubiquitination serves as a recognition signal for autophagic degradation of misfolded proteins and damaged organelles.⁴The accumulation of damaged organelles and protein aggregates are believed to be harmful to the cells and cannot be efficiently removed by the proteasomes.^{4, 5, 6} Our previous studies have demonstrated an aberrant accumulation of misfolded/ubiquitin protein aggregates in coxsackievirus-infected cells and mouse hearts, suggesting that defective protein degradation may have a role in viral pathogenesis.^{7, 8, 9} Coxsackievirus type B3 (CVB3), an enterovirus belonging to the family Picornaviridae, is a common cause of viral myocarditis and several other diseases in human.¹⁰ We have recently demonstrated that autophagy adapter protein SQSTM1 is cleaved through the proteolytic activity of viral protease 2A^pro.¹¹ The cleavage of SQSTM1 results in the dissociation of the N-terminal PB1 domain from the C-terminal UBA and LIR domains, and subsequently the loss-of-function of full-length SQSTM1 in mediating selective autophagy.¹¹NBR1 is a functional homolog of SQSTM1. Despite the difference of its primary sequence and size from those of SQSTM1, NBR1 shares a similar domain structure with SQSTM1, both containing PB1, ZZ (zinc-finger domain), LIR and UBA domains.¹² Depending on the characteristics of the substrates, NBR1 and SQSTM1 work either independently or cooperatively with each other. It was reported that degradation of bacteria, such as Salmonella and Listeria, requires SQSTM1, but not NBR1.¹³ In contrast, NBR1 alone is sufficient to target peroxisomes to lysosomes for degradation in the absence of SQSTM1.¹⁴ NBR1 can also work in concert with SQSTM1 to remove ubiquitinated cargoes.¹² The observation that deletion of SQSTM1 fails to induce the accumulation of ubiquitinated proteins implies a possible compensatory role for NBR1 in the compromise of SQSTM1 function.^{3, 15}The purpose of this study was to determine whether NBR1 could compensate for disrupted SQSTM1 function following coxsackievirus infection. Here we showed that NBR1 was also cleaved during CVB3 infection by virus-encoded protease 2A^pro and 3C^pro. We further provided evidence that cleavage of both SQSTM1 and NBR1 not only led to the loss of their native function, but also resulted in the generation of cleavage mutants that display dominant-negative activities. Finally, we demonstrated a mutual regulatory relationship between SQSTM1 and NBR1 during viral infection. Our data suggest a new mechanism through which coxsackievirus infection induces abnormal accumulation of ubiquitin conjugates.     

Cell Fate Decisions in Malignant Hematopoiesis: Leukemia Phenotype Is Determined by Distinct Functional Domains of the MN1 Oncogene

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.     

Strategies for Tracking Anastasis,A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for “rising to life”) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor for identifying and tracking anastasis in mammalian cells.     

MAD2 expression and its significance in mitotic checkpoint control in testicular germ cell tumour

Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p<0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p=0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.