Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.     

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Engineering therapeutic protein disaggregases

Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD.We urgently need to pioneer game-changing solutions to remedy a number of increasingly prevalent and fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD; Cushman et al., 2010 ; Jackrel and Shorter, 2015 ). These disorders relentlessly erode our morale and economic resources. Aging is the major risk factor for all of these diseases, which threaten public health on a global scale and represent a severe impediment to living longer lives. A number of promising drugs have emerged to treat cancer and heart disease, but, distressingly, this is not the case for these and other neurodegenerative diseases, for which drug pipelines lie dormant and empty. This situation is unacceptable, and an impending healthcare crisis looms worldwide as population demographics inexorably shift toward older age groups.ALS, PD, AD, and related neurodegenerative disorders are unified by a common underlying theme: the misfolding and aggregation of specific proteins (characteristic for each disease) in the CNS (Cushman et al., 2010 ; Eisele et al., 2015 ). Thus, in ALS, typically an RNA-binding protein with a prion-like domain, TDP-43, mislocalizes from the nucleus to cytoplasmic inclusions in degenerating motor neurons (Neumann et al., 2006 ; Gitler and Shorter, 2011 ; King et al., 2012 ; Robberecht and Philips, 2013 ; March et al., 2016 ). In PD, α-synuclein forms toxic soluble oligomers and cytoplasmic aggregates, termed Lewy bodies, in degenerating dopaminergic neurons (Dehay et al., 2015 ). By contrast, in AD, amyloid-β (Aβ) peptides form extracellular plaques and the microtubule-binding protein, tau, forms cytoplasmic neurofibrillary tangles in afflicted brain regions (Jucker and Walker, 2011 ). Typically, these disorders are categorized into ∼80–90% sporadic cases and ∼10–20% familial cases. Familial forms of disease often have clear genetic causes, which might one day be amenable to gene editing via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 therapeutics if critical safety and ethical concerns can be successfully addressed and respected (Doudna and Charpentier, 2014 ; Baltimore et al., 2015 ; Rahdar et al., 2015 ; Callaway, 2016 ). However, the more common sporadic forms of disease often have no clear underlying genetics, and wild-type proteins misfold (Cushman et al., 2010 ; Jucker and Walker, 2011 ; Robberecht and Philips, 2013 ; Dehay et al., 2015 ). Consequently, therapeutic agents that directly target and safely reverse deleterious protein misfolding are likely to have broad utility (Eisele et al., 2015 ).There are no treatments that directly target the reversal of the protein-misfolding phenomena that underlie these disorders (Jackrel and Shorter, 2015 ). Strategies that directly reverse protein misfolding and restore proteins to native form and function could, in principle, eradicate any severely damaging loss-of-function or toxic gain-of-function phenotypes caused by misfolded conformers (Figure 1; Jackrel and Shorter, 2015 ). Moreover, therapeutic disaggregases would dismantle self-templating amyloid or prion structures, which spread pathology and nucleate formation of neurotoxic, soluble oligomers (Figure 1; Cushman et al., 2010 ; Cohen et al., 2013 ; Guo and Lee, 2014 ; Jackrel and Shorter, 2015 ). My group has endeavored to engineer and evolve Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast (DeSantis and Shorter, 2012 ; Sweeny and Shorter, 2015 ), to more effectively disaggregate misfolded proteins connected with various neurodegenerative disorders, including ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). Although wild-type Hsp104 can disaggregate diverse amyloid and prion conformers, as well as toxic soluble oligomers (Lo Bianco et al., 2008 ; DeSantis et al., 2012 ), its activity against human neurodegenerative disease proteins is suboptimal. Is it even possible to improve on existing Hsp104 disaggregase activity, which has been wrought over the course of millions of years of evolution?Open in a separate windowFIGURE 1:Therapeutic protein disaggregases. Two malicious problems are commonly associated with protein misfolding into disordered aggregates, toxic oligomers, and cross–β amyloid or prion fibrils: 1) a toxic gain of function of the protein in various misfolded states; and 2) a loss of function of the protein in the various misfolded states. These problems can contribute to the etiology of diverse neurodegenerative diseases in a combinatorial or mutually exclusive manner. A therapeutic protein disaggregase would reverse protein misfolding and recover natively folded functional proteins from disordered aggregates, toxic oligomers, and cross–β amyloid or prion fibrils. In this way, any toxic gain of function or toxic loss of function caused by protein misfolding would be simultaneously reversed. Ideally, all toxic misfolded conformers would be purged. Therapeutic protein disaggregases could thus have broad utility for various fatal neurodegenerative diseases.Remarkably, the answer to this question is yes! We used nimble yeast models of neurodegenerative proteinopathies (Outeiro and Lindquist, 2003 ; Gitler, 2008 ; Johnson et al., 2008 ; Sun et al., 2011 ; Khurana et al., 2015 ) as a platform to isolate enhanced disaggregases from large libraries of Hsp104 variants generated by error-prone PCR (Jackrel et al., 2014b ). In this way, we reprogrammed Hsp104 to yield the first disaggregases that reverse TDP-43, FUS (another RNA-binding protein with a prion-like domain connected to ALS), and α-synuclein (connected to PD) aggregation and proteotoxicity (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ; Torrente et al., 2016 ). Remarkably, a therapeutic gain of Hsp104 function could be elicited by a single missense mutation (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Under conditions in which Hsp104 was ineffective, potentiated Hsp104 variants dissolved protein inclusions, restored protein localization (e.g., TDP-43 returned to the nucleus from cytoplasmic inclusions), suppressed proteotoxicity, and attenuated dopaminergic neurodegeneration in a Caenorhabditis elegans PD model (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Remarkably, these therapeutic modalities originated from degenerate loss of amino acid identity at select positions of Hsp104 rather than specific mutation (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Some of these changes were remarkably small (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Thus, potentiated Hsp104 variants could be generated by removal of a methyl group from a single side chain or addition or removal of a single methylene bridge from a single side chain (Jackrel et al., 2014a , 2015 ; Jackrel and Shorter, 2015 ). Thus, small molecules that bind in accessible regions of Hsp104 rich in potentiating mutations might also be able to enhance activity. However, a small-scale screen for small-molecule modulators of Hsp104 revealed only inhibitors (Torrente et al., 2014 ). Nonetheless, our work has established that disease-associated aggregates and amyloid are tractable targets and that enhanced artificial disaggregases can restore proteostasis and mitigate neurodegeneration (Jackrel and Shorter, 2015 ).One surprising aspect of this work is just how many Hsp104 variants we could isolate with potentiated activity. We now have hundreds (Jackrel et al., 2014a ; Jackrel et al., 2015 ). Typically, potentiated Hsp104 variants displayed enhanced activity against several neurodegenerative disease proteins. For example, Hsp104^A503S rescued the aggregation and toxicity of TDP-43, FUS, TAF15, and α-synuclein (Jackrel et al., 2014a ; Jackrel and Shorter, 2014 ). By contrast, some potentiated Hsp104 variants rescued only the aggregation and toxicity of a subset of disease proteins. For example, Hsp104^D498V rescued only the aggregation and toxicity of FUS and α-synuclein (Jackrel et al., 2014a ). A challenge that lies ahead is to engineer potentiated Hsp104 variants that are highly substrate specific to mitigate any potential off-target effects, should they arise (Jackrel and Shorter, 2015 ).Very small changes in primary sequence yield potentiated Hsp104 variants. However, Hsp104 has no metazoan homologue (Erives and Fassler, 2015 ). Now comes the important point. Neuroprotection could be broadly achieved by making very subtle modifications to existing human chaperones with newly appreciated disaggregase activity—for example, Hsp110, Hsp70, and Hsp40 (Torrente and Shorter, 2013 ) and HtrA1 (Poepsel et al., 2015 ).Whether Metazoa even possess a powerful protein disaggregation and reactivation machinery had been a long-standing enigma (Torrente and Shorter, 2013 ). However, it has recently emerged that two metazoan chaperone systems—1) Hsp110, Hsp70, and Hsp40 (Torrente and Shorter, 2013 ) and 2) HtrA1 (Poepsel et al., 2015 )—possess disaggregase activity that could be therapeutically harnessed or stimulated to reverse deleterious protein misfolding in neurodegenerative disease. I suspect that Metazoa harbor additional disaggregase systems that remain to be identified (Guo et al., 2014 ). Whether due to vicissitudes of aging, environment, or genetic background, these disaggregase systems fail in the context of ALS, PD, and AD. Based on the surprising precedent of our potentiated versions of Hsp104 (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ), I hypothesize that it is possible to engineer and evolve potentiated variants of these human protein disaggregases to more effectively counter deleterious misfolding events in ALS, PD, and AD (Torrente and Shorter, 2013 ; Mack and Shorter, 2016 ).Using classical biochemical reconstitution, it was discovered that one mammalian protein-disaggregase system comprises three molecular chaperones—Hsp110, Hsp70, and Hsp40—which synergize to dissolve and reactivate model proteins trapped in disordered aggregates and can even depolymerize amyloid fibrils formed by α-synuclein from their ends (Shorter, 2011 ; Duennwald et al., 2012 ; Torrente and Shorter, 2013 ). Hsp110, Hsp70, and Hsp40 isoforms are found in the cytoplasm, nucleus, and endoplasmic reticulum, which suggest that protein disaggregation and reactivation can occur in several compartments (Finka et al., 2015 ). Subsequent studies suggest that this system may be more powerful than initially anticipated (Rampelt et al., 2012 ; Mattoo et al., 2013 ; Gao et al., 2015 ; Nillegoda et al., 2015 ). Nonetheless, this system must become overwhelmed in neurodegenerative disorders. Perhaps selectively vulnerable neurons display particular deficits in this machinery. Hence, potentiating the activity of this system via engineering could be extremely valuable. It is promising that directed evolution studies yielded DnaK (Hsp70 in Escherichia coli) variants with improved ability to refold specific substrates (Aponte et al., 2010 ; Schweizer et al., 2011 ; Mack and Shorter, 2016 ), but whether this can be extended to human Hsp70 and the disaggregation of neurodegenerative disease proteins is uncertain.It is exciting that recent studies have revealed that HtrA1, a homo-oligomeric PDZ serine protease, can dissolve and degrade AD-linked tau and Aβ42 fibrils in an ATP-independent manner (Tennstaedt et al., 2012 ; Poepsel et al., 2015 ). HtrA1 first dissolves tau and Aβ42 fibrils and then degrades them, as protease-defective HtrA1 variants dissolve fibrils without degrading them (Poepsel et al., 2015 ). HtrA1 is found in the cytoplasm (∼30%) but is also secreted (∼70%; Poepsel et al., 2015 ). Indeed, HtrA1 is known to degrade substrates in both the extracellular space and the cytoplasm (Chien et al., 2009 ; Campioni et al., 2010 ; Tiaden and Richards, 2013 ). Thus HtrA1 could dissolve Aβ42 fibrils in the extracellular space and tau fibrils in the cytoplasm and simultaneously destroy the two cardinal features of AD (Poepsel et al., 2015 ). I suspect that this system becomes overwhelmed or is insufficient in AD, and thus potentiating and tailoring HtrA1 disaggregase activity could be a valuable therapeutic strategy. For example, it might be advantageous to simply degrade Aβ42 after disaggregation if the peptide has no beneficial function. Thus HtrA1 variants with enhanced disaggregation and degradation activity against Aβ42 could be extremely useful. However, Aβ42 (and related Aβ peptides) may have physiological functions that are presently underappreciated (Soscia et al., 2010 ; Fedele et al., 2015 ), in which case HtrA1 variants with enhanced disaggregase activity but reduced proteolytic activity could be vital. HtrA1 variants with enhanced disaggregase activity but reduced proteolytic activity may also be particularly important to recover functional tau from neurofibrillary tangles to reverse loss of tau function in AD and various tauopathies (Santacruz et al., 2005 ; Trojanowski and Lee, 2005 ; Dixit et al., 2008 ).I suggest that relatively small changes in primary sequence will yield large increases in disaggregase activity for these systems as they do for Hsp104 (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). If true, this would further suggest that small molecules that bind in the appropriate regions of Hsp110, Hsp70, Hsp40, or HtrA1 might also enhance disaggregase activity. Thus, isolating small-molecule enhancers of the Hsp110, Hsp70, and Hsp40 or HtrA1 disaggregase systems could yield important therapeutics. Indeed, I hypothesize that enhancing the activity of the Hsp110, Hsp70, and Hsp40 or HtrA1 disaggregase system with specific small molecules will enable dissolution of toxic oligomeric and amyloid forms of various disease proteins and confer therapeutic benefits in ALS, PD, AD, and potentially other neurodegenerative disorders.It is intriguing that several small molecules are already known to enhance various aspects of Hsp70 chaperone activity (Pratt et al., 2015 ; Shrestha et al., 2016 ). These include MKT-077, JG-98, YM-1, YM-8, and 115-7c (Wisen et al., 2010 ; Pratt et al., 2015 ). It is not known whether any of these stimulates the disaggregase activity of the Hsp110, Hsp70, and Hsp40 system. MKT-077, JG-98, YM-1, and YM-8 are rhodocyanines that bind with low micromolar affinity to the nucleotide-binding domain of ADP- but not ATP-bound Hsp70, stabilizing the ADP-bound state (Pratt et al., 2015 ). The ADP-bound state of Hsp70 engages clients with higher affinity, and consequently MKT-077, JG-98, and YM-1 activate binding of Hsp70 to misfolded proteins (Wang et al., 2013 ; Pratt et al., 2015 ). Thus, under some conditions, these small molecules can promote folding of certain Hsp70 clients (Morishima et al., 2011 ; Pratt et al., 2015 ). However, prolonged interaction of clients with Hsp70 promotes their CHIP-dependent ubiquitylation and degradation in vivo (Morishima et al., 2011 ; Wang et al., 2013 ; Pratt et al., 2015 ). Intriguingly, YM-1 promotes clearance of polyglutamine oligomers and aggregates in cells (Wang et al., 2013 ; Pratt et al., 2015 ). MKT-0777, YM-1, JG-98, and YM-8 also promote clearance of tau and confer therapeutic benefit in tauopathy models (Abisambra et al., 2013 ; Miyata et al., 2013 ; Fontaine et al., 2015 ). Of importance, YM-8 is long lived in vivo and crosses the blood–brain barrier (Miyata et al., 2013 ). The dihydropyrimidine 115-7c activates Hsp70 ATPase turnover rate, promotes Hsp70 substrate refolding, and reduces α-synuclein aggregation in cell culture (Wisen et al., 2010 ; Kilpatrick et al., 2013 ). It binds to the IIA subdomain of Hsp70 and promotes the active Hsp70–Hsp40 complex (Wisen et al., 2010 ). Small-molecule enhancers of HtrA1 protease activity have also emerged (Jo et al., 2014 ). Thus it will be important to assess whether these small molecules enhance the activity of their respective disaggregases against various neurodegenerative substrates.Although small molecules that enhance disaggregase activity of endogenous human proteins might be the most immediately translatable, gene-, mRNA-, or protein-based therapies can also be envisioned. For example, adeno-associated viruses expressing enhanced disaggregases might be used to target degenerating neurons (Dong et al., 2005 ; Lo Bianco et al., 2008 ; Deverman et al., 2016 ). Alternatively, if viral vectors are undesirable, modified mRNAs might serve as an alternative to DNA-based gene therapy (Kormann et al., 2011 ). Protein-based therapeutics could also be explored. For example, intraperitoneal injection of human Hsp70 increased lifespan, delayed symptom onset, preserved motor function, and prolonged motor neuron viability in a mouse model of ALS (Gifondorwa et al., 2007 ; Gifondorwa et al., 2012 ). Several other studies suggest that exogenous delivery of Hsp70 can have beneficial, neuroprotective effects in mice (Nagel et al., 2008 ; Bobkova et al., 2014 ; Bobkova et al., 2015 ).Ultimately, if safety and ethical concerns can be overcome in a circumspect, risk-averse manner, CRISPR-Cas9–based therapeutics might even be used to genetically alter the underlying disaggregase to a potentiated form in selectively vulnerable neuronal populations. This approach might be particularly valuable if enhanced disaggregase activity is not detrimental in the long term. Moreover, stem cell–based therapies for replacing lost neurons could also be fortified to express enhanced disaggregase systems. Thus they would be endowed with resistance to potential infection by prion-like conformers that might have accumulated during disease progression (Cushman et al., 2010 ).Enhanced disaggregase activity is likely to be highly advantageous to neurons under circumstances in which protein misfolding has overwhelmed the system (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). However, inappropriate hyperactivity of protein disaggregases might also have detrimental, off-target effects under regular conditions in which protein misfolding is not an overwhelming issue (Jackrel et al., 2014a ; Jackrel and Shorter, 2015 ). Thus it may be advantageous to engineer enhanced protein disaggregases to be highly substrate specific. In this way, off-target effects would be readily avoided. There is strong precedent for directed evolution or engineering of specialized chaperone or protein activity from a generalist antecedent (Wang et al., 2002 ; Farrell et al., 2007 ; Smith et al., 2015 ). Thus, engineering specialist disaggregases for each disease substrate could be achieved. Alternatively, transient or intermittent doses of enhanced disaggregases at specific times or places where they are most needed would also minimize potentially toxic side effects. For example, enhanced disaggregase activity might be applied ephemerally to clear existing misfolded conformers and then be withdrawn once the endogenous proteostasis network regains control. Similarly, it is straightforward to envision administration of small-molecule enhancers of disaggregase activity in intermittent protocols that enable facile recovery from potential side effects (Fontaine et al., 2015 ). In this way, any adverse effects of enhanced protein-disaggregase activity under normal physiological conditions would be avoided. Many barriers will need to be safely overcome to implement a successful therapeutic disaggregase, including how to deliver enhanced disaggregase activity to exactly where it is needed. However, these obstacles are not a reason to be pessimistic. On the contrary, the isolation of engineered disaggregases that efficaciously reverse deleterious misfolding of neurodegenerative disease proteins directs our attention to considerably expand the environs in which they should be sought. My closing sentences, therefore, are intended to be provocative.I suspect that neuroprotection could be broadly actualized via precise but subtle alterations to existing protein-disaggregase modalities. The engineering and evolution of protein disaggregases could yield important solutions to avert an imminent plague of neurodegenerative disorders that promises to devastate our society. I strongly suspect that cures for various neurodegenerative disorders will be realized by pioneering as-yet-uncharted regions of disaggregase sequence space or chemical space to elucidate small-molecule enhancers of disaggregase activity.     

Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica

Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in E. histolytica.Pyridine nucleotide transhydrogenase (PNT) participates in the bioenergetic processes of the cell. PNT generally resides on the cytoplasmic membranes of bacteria and the inner membrane of mammalian mitochondria (3, 16) and utilizes the electrochemical proton gradient across the membrane to drive NADPH formation from NADH (14, 15, 39) according to the reaction H⁺out + NADH + NADP⁺↔H⁺in + NAD⁺ + NADPH, where “out” and “in” denote the cytosol and the matrix of the mitochondria, or the periplasmic space and the cytosol of bacteria, respectively.PNT has been identified in several protozoan parasites, including Entamoeba histolytica (8, 51), Eimeria tenella (17, 47), Mastigamoeba balamuthi (11) Plasmodium falciparum (10), Plasmodium yoelii (6), and Plasmodium berghei (12). In general, PNT contains conserved structural units consisting of three domains, the NAD(H)-binding domain (domain I [dI]) and the NADP(H)-binding domain (domain III [dIII]), both of which face the matrix side of the eukaryotic mitochondria or the cytoplasmic side in bacteria, and the hydrophobic domain (domain II [dII]), containing 11 to 13 transmembrane regions. PNT from E. tenella and E. histolytica exists as a single polypeptide in an unusual configuration consisting of dIIb-dIII-dI-dIIa, with a 38-amino-acid-long linker region between dIII and dI (48).E. histolytica, previously considered an “amitochondriate” protist, is currently considered to possess a mitochondrion-related organelle with reduced and divergent functions, the mitosome (1, 21, 23a, 26, 42). Our recent proteomic study of isolated mitosomes identified about 20 new constituents (26), together with four proteins previously demonstrated in E. histolytica mitosomes: Cpn60 (8, 19, 21, 42), Cpn10 (46), mitochondrial Hsp70 (2, 44), and mitochondrion carrier family (MCF) (ADP/ATP transporter) (7). Despite the early presumption of PNT being localized in mitosomes (8), based on the amino-terminal region rich in hydroxylated (five serines and threonines) and acidic (three glutamates) amino acids, which slightly resembles known mitochondrion- and hydrogenosome-targeting sequences (8, 35), PNT was not discovered in the mitosome proteome. We also doubted this premise because PNT was one of the major proteins identified in isolated phagosomes (32, 33). Thus, the intracellular localization and trafficking of PNT remain unknown.In this report, we showed that E. histolytica PNT (EhPNT) is localized to various vesicles and vacuoles, including lysosomes and phagosomes, using wild-type amoebae and antiserum raised against recombinant EhPNT and an E. histolytica line expressing EhPNT with a carboxyl-terminal hemagglutinin (HA) epitope tag and anti-HA antibody. We also showed that all domains of EhPNT are required for its trafficking to the acidic compartment by using amoeba transformants expressing the HA tag or green fluorescent protein (GFP) fused with a region containing various domains of EhPNT.     

Three-dimensional Structure of Acanthamoeba castellanii Myosin-IB (MIB) Determined by Cryoelectron Microscopy of Decorated Actin Filaments

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a “classical” myosin-I, Acanthamoeba myosin-IB (MIB), at ∼18 Å resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, ∼10°, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.The myosin superfamily consists of at least 12 distinct classes that vary both in the sequence of their conserved myosin catalytic domains as well as in their unique tails (Mooseker and Cheney, 1995; Sellers and Goodson, 1995). For many years the only known myosins were the double-headed, filament-forming myosins found in muscle (conventional myosins or myosins-II). The remaining classes of myosin have been termed “unconventional myosins” to differentiate them from the myosins-II. Probably the most thoroughly studied class of unconventional myosins is the myosin-I class. These small, single-headed myosins bind to membrane lipids through a basic domain in their tail (for review see Pollard et al., 1991; Mooseker and Cheney, 1995). The first unconventional myosin (and first myosin-I) was isolated from Acanthamoeba castellanii (Pollard and Korn, 1973 a,b), and was purified on the basis of its K⁺, EDTA, and actin-activated MgATPase activities. However, this myosin was unusual in that it had a single heavy chain of ∼140 kD, in contrast to the two ∼200-kD heavy chains of myosin-II (Pollard and Korn, 1973 a).Three isoforms of the classical Acanthamoeba myosins-I are now known: myosins-IA, -IB, and -IC (Maruta and Korn, 1977a ,b; Maruta et al., 1979). Each of the isoforms consists of a conserved myosin catalytic domain, a binding site for one or two light chains, a basic domain, a GPA¹(Q) domain (rich in glycine, proline and alanine [glutamine]), and an scr-homology domain-3 (SH3) domain (Pollard et al., 1991; Mooseker and Cheney, 1995). These myosins-I can associate with membranes or with actin filaments through their tail domains. An electrostatic association of myosin-I with anionic phospholipids and with base-stripped membranes has been shown to occur (Adams and Pollard, 1989; Miyata et al., 1989; Hayden et al., 1990), and this interaction has been mapped to the basic domain (Doberstein and Pollard, 1992). Interestingly, these myosins also contain a second, ATP-insensitive actin binding site (Lynch et al., 1986) enabling them to mediate actin–actin movements (Albanesi et al., 1985; Fujisaki et al., 1985). In myosin-IA (Lynch et al., 1986) and myosin-IC (Doberstein and Pollard, 1992), this binding site was localized to the GPA domain. Acanthamoeba myosins-I have maximal steady-state actin-activated ATPase rates of ∼10–20 s⁻¹ (Pollard and Korn, 1973 b; Albanesi et al., 1983), and an unusual triphasic dependence upon actin concentration (Pollard and Korn, 1973 b; Albanesi et al., 1983). This triphasic activation is due to the actin cross-linking ability imparted by the ATP-insensitive actin binding site on the tail (Albanesi et al., 1985). Analysis of the individual steps in the ATPase cycle by transient kinetics revealed that the mechanism of myosin-IA is similar to slow skeletal muscle myosin, whereas myosin-IB (MIB) is similar to fast skeletal muscle myosin (Ostap and Pollard, 1996). The in vitro motility of myosin-I has also been well characterized (Zot et al., 1992). The maximal rate of filament sliding is ∼0.2 μm s⁻¹. Interestingly, this rate is ∼10–50× slower than the rates observed for skeletal muscle myosin, even though the ATPase rates are comparable.MIB consists of a 125-kD heavy chain and a single 27-kD light chain (Maruta et al., 1979; Jung et al., 1987). This isoform is primarily associated with the plasma membrane as well as vacuolar membranes (Baines et al., 1992). MIB appears to be associated with the plasma membrane at sites of phagocytosis and was concentrated at the tips of pseudopodia (Baines et al., 1992). This localization suggests that MIB may be involved in membrane dynamics at the cell surface. MIB is regulated by heavy chain phosphorylation of serine 411 (Brzeska et al., 1989, 1990), which is located at the actin-binding site (Rayment et al., 1993). Similar to the myosin-I isoforms in Acanthamoeba, heavy chain phosphorylation results in >20-fold activation of the actin-activated myosin-I ATPase activity (Albanesi et al., 1983). This activation is not the result of changes in the binding of myosin-I to F-actin (Albanesi et al., 1983; Ostap and Pollard, 1996). The transient kinetic studies of Ostap and Pollard (1996) suggest that phosphorylation regulates the rate-limiting phosphate release step, the transition from weakly bound intermediates in rapid equilibrium with actin to strongly bound states, capable of sustaining force.Despite the extensive analysis of ameboid myosin-I biochemical properties and in vivo function, there is little structural information on these myosins. The only detailed structural information available for the myosins-I comes from recent electron microscopy studies on brush border myosin-I (BBM-I) (Jontes et al., 1995; Jontes and Milligan, 1997a ,b; Whittaker and Milligan, 1997), a structurally distinct myosin-I subtype. Therefore, we investigated the structure of a “classical,” ameboid-type myosin, Acanthamoeba MIB using electron microscopy. First, electron micrographs of negatively stained two-dimensional (2D) crystals were used to generate a projection map of MIB at ∼18 Å resolution. In addition, we used cryoelectron microscopy and helical image analysis to produce a moderate resolution three-dimensional (3D) map (30 Å) of actin filaments decorated with MIB. These studies enabled us to compare the structure of MIB with BBM-I. The comparison of MIB with BBM-I reveals marked structural differences in the tail domains of these two proteins; MIB appears to have a much shorter “lever arm” and a more compact tail, whereas most of the BBM-I mass is composed of an extended light chain–binding domain (LCBD). In addition, the MIB catalytic domain appears to be slightly tilted compared to BBM-I, with respect to the F-actin axis. Our structural results suggest that these two types of myosin-I may have distinct intracellular functions.     

Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA.Germinating asexual spores (conidiospores) of Aspergillus nidulans transiently undergo isotropic growth (“swelling”) before establishing a polarity axis that grows by apical extension, leading to the characteristic tubular morphology of the fungal cell (15, 16, 33). Stable maintenance of a polarity axis at the high apical extension rates of A. nidulans (∼0.5 μm/min at 25°C) (23) can be attributable, at least in part, to the polarization of the secretory apparatus and the predominant and highly efficient delivery of secretory vesicles to the apex (8, 18, 40, 49). In addition, work from several laboratories strongly indicated that hyphal tip growth also involves endocytosis. A key observation supporting this involvement was that despite the fact that endocytosis can occur elsewhere, the endocytic internalization machinery predominates in the hyphal tip, forming a subapical collar. The spatial association of this collar with the apical region where secretory materials are delivered would allow removal of excess lipids/proteins reaching the plasma membrane with secretory vesicles (1, 2, 30, 49, 51, 57), but, most importantly, rapid endocytic recycling (i.e., efficient endocytosis of membrane proteins followed by their redelivery to the plasma membrane) can generate and maintain polarity, as shown with the v-SNARE and secretory-vesicle-resident SynA, which is a substrate of the subapical endocytic ring (1, 49, 52). It is plausible that such a mechanism could drive the polarization of one or more proteins acting as positional cues to mediate polarity maintenance.Genetic evidence strongly supported the conclusion that endocytosis is required for apical extension. Mutational inactivation of the A. nidulans fimbrin FimA or of the small GTPase ArfB^Arf6 (a regulator of endocytosis from fungi to mammals), led to delayed polarity establishment and morphologically aberrant tips indicative of polarity maintenance defects (30, 51). These mutations, although very severely debilitating, are not lethal. In contrast, heterokaryon rescue showed that SlaB, a key F-actin regulator of the endocytic internalization machinery (26), is essential in A. nidulans (2). slaBΔ cells are able to establish polarity, but they arrest in apical extension during the very early steps of polarity maintenance with a bud-like germ tube (2). However, work with Aspergillus oryzae using a thiamine-repressible promoter to drive A. oryzae End4 (AoEnd4) (SlaB) expression showed that although endocytosis was prevented and hyphal morphology altered under repressing conditions, hyphal tip extension and polarity maintenance were not completely abolished (20), perhaps suggesting that the phenotype of the thiamine-repressed conditional allele might not fully resemble the null phenotype.F-actin strongly predominates in the hyphal tips (2, 14, 17, 49, 51). Because endocytic internalization is powered by F-actin (27), predominance of endocytic “patches” (i.e., sites of endocytic internalization) in the tip accounts, at least in part, for F-actin polarization. However, F-actin plays fundamental nonendocytic roles in the tip; for example, actin cables nucleated by the SepA formin localizing to the apical crescent are thought to play a major role in secretion, whereas a network of F-actin microfilaments appears to be a major component of the Spitzenkörper (4, 21, 43, 49). Notably, all genes used to address the role of endocytosis in apical extension share in common their involvement in regulating F-actin. Thus, the Saccharomyces cerevisiae ArfB orthologue Arf3p regulates endocytosis but also appears to regulate F-actin at multiple levels (12, 28, 44). Like their respective S. cerevisiae orthologues Sla2p and Sac6p, SlaB and FimA are key components of endocytic patches, but in budding yeast their orthologues appear to regulate F-actin dynamics beyond endocytosis (27, 35, 56).To gain insight into the essential role of SlaB in A. nidulans, we designed a conditional expression allele. Time-lapse microscopy under restrictive conditions demonstrated that polarity establishment is essentially normal but that these mutant germ tubes arrested in apical extension subsequently undergo swelling, acquiring the characteristic bud-like shape of abortive slaBΔ germlings. Medium shift experiments allowed us to address the role of SlaB in apical extension beyond these early stages of polarity maintenance. We demonstrate the key role that SlaB plays in endocytosis and polarity maintenance, but we also show that deficiency of SlaB affects the actin cytoskeleton.     

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin^ΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.Tight junctions seal the paracellular space in simple epithelia, such as those lining the lungs, intestines, and kidneys (Anderson et al., 2004 ; Fanning and Anderson, 2009 ; Shen et al., 2011 ). In the intestine, reduced paracellular barrier function is associated with disorders in which increased paracellular flux of ions and molecules contributes to symptoms such as diarrhea, malabsorption, and intestinal protein loss. Recombinant tumor necrosis factor (TNF) can be used to model this barrier loss in vitro or in vivo (Taylor et al., 1998 ; Clayburgh et al., 2006 ), and TNF neutralization is associated with restoration of intestinal barrier function in Crohn''s disease (Suenaert et al., 2002 ). Further, in vivo and in vitro studies of intestinal epithelia show that TNF-induced barrier loss requires myosin light chain kinase (MLCK) activation (Zolotarevsky et al., 2002 ; Clayburgh et al., 2005 , 2006 ; Ma et al., 2005 ; Wang et al., 2005 ). The resulting myosin II regulatory light chain (MLC) phosphorylation drives occludin internalization, which is required for cytokine-induced intestinal epithelial barrier loss (Clayburgh et al., 2005 , 2006 ; Schwarz et al., 2007 ; Marchiando et al., 2010 ). In addition, transgenic EGFP-occludin expression in vivo limits TNF-induced depletion of tight junction–associated occludin, barrier loss, and diarrhea (Marchiando et al., 2010 ). Conversely, in vitro studies show that occludin knockdown limits TNF-induced barrier regulation (Van Itallie et al., 2010 ). The basis for this discrepancy is not understood.One challenge is that, despite being identified 20 yr ago (Furuse et al., 1993 ), the contribution of occludin to tight junction regulation remains incompletely defined. The observation that occludin-knockout mice are able to form paracellular barriers and do not have obvious defects in epidermal, respiratory, or bladder tight junction function (Saitou et al., 2000 ; Schulzke et al., 2005 ) led many to conclude that occludin is not essential for tight junction barrier function. It is important to note, however, that barrier regulation in response to stress has not been studied in occludin-deficient animals.We recently showed that dephosphorylation of occludin serine-408 promotes assembly of a complex composed of occludin, ZO-1, and claudin-2 that inhibits flux across size- and charge-selective channels termed the pore pathway (Anderson and Van Itallie, 2009 ; Turner, 2009 ; Raleigh et al., 2011 ; Shen et al., 2011 ). Although this demonstrates that occludin can serve a regulatory role, it does not explain the role of occludin in TNF-induced barrier loss, which increases flux across the size- and charge-nonselective leak pathway (Wang et al., 2005 ; Weber et al., 2010 ). In vitro studies, however, do suggest that occludin contributes to leak pathway regulation, as occludin knockdown in either Madin–Darby canine kidney (MDCK) or human intestinal (Caco-2) epithelial monolayers enhances leak pathway permeability (Yu et al., 2005 ; Al-Sadi et al., 2011 ; Ye et al., 2011 ). Taken as a whole, these data suggest that occludin organizes the tight junction to limit leak pathway flux, whereas occludin removal, either by knockdown or endocytosis, enhances leak pathway flux.To define the mechanisms by which occludin regulates the leak pathway, we analyzed the contributions of occludin, as well as specific occludin domains, to basal and TNF-induced barrier regulation. The data indicate that TNF destabilizes tight junction–associated occludin via interactions mediated by the C-terminal coiled-coil occludin/ELL domain (OCEL). Further, these OCEL-mediated events are required for TNF-induced barrier regulation. Thus these data provide new insight into the structural elements and mechanisms by which occludin regulates leak pathway paracellular flux.     

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. The host immune response toward aerosol infection is to quarantine tubercle bacilli in a granulomatous structure (1, 2). However, granuloma-associated mycobacteria can switch to a non-replicative, “dormant” state and successfully evade immune response for decades after infection (3, 4). The metabolic events that permit tubercle bacilli to enter host cells and revive from states of persistence suggest that lipids are utilized as a carbon source (5–7). During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG)¹-enriched lipid droplets (8–10). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.Comparative sequence analysis of the Mtb genome has revealed that it contains 250 genes encoding enzymes involved in lipid metabolism compared with only 50 enzymes in Escherichia coli, which has a genome of comparable size. Among these genes, 150 are predicted to encode proteins involved in lipid catabolism (12, 13). A family of 24 carboxyl ester hydrolases called “lip” genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15–18).Tetrahydrolipstatin, a serine esterase inhibitor, covalently binds to and inhibits mammalian lipases and fatty acid synthase (FAS) and is marketed as “Orlistat” for the treatment of severe forms of obesity (19). THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20–22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23–25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria. Importantly, we systematically compared readouts of fluorescently tagged THL-proteins (7 bands on one-dimensional SDS-PAGE) with those of mass spectrometry-based peptide identification of enriched protein fractions (247 in growing cells). This comparison led to the identification of 14 THL targets, two of which were further validated experimentally. We furthermore provide a conceptual framework for the evaluation of this target list using both experimental as well as bioinformatics approaches in two examples, lipH and tesA. Overall, our data indicate that THL is an anti-mycobacterial drug because of its potential to (1) bind to a relatively wide range of lipolytic enzymes and (2) prevent bacilli from resuscitating from a nonreplicating persistent (NRP) state when lipid metabolism is particularly important.     

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn''s disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.     

Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.Candida albicans is a major opportunistic fungal human pathogen that causes a wide variety of infections (9, 68). In healthy individuals C. albicans resides as a commensal within the oral cavity and gastrointestinal and urogenital tracts. However, in immunocompromised hosts, C. albicans causes infections ranging in severity from mucocutaneous infections to life-threatening disseminated diseases (9, 68). Research into the pathogenicity of C. albicans has revealed a complex mix of putative virulence factors (7, 60), perhaps reflecting the fine balance this species strikes between commensal colonization and opportunistic invasion of the human host.Melanins are biological pigments, typically dark brown or black, formed by the oxidative polymerization of phenolic compounds. They are negatively charged hydrophobic molecules with high molecular weights and are insoluble in both aqueous and organic solvents. Their insolubility makes melanins difficult to study, and no definitive structure has yet been found for them; they probably represent an amorphous mixture of polymers (35). There are various types of melanin in nature, including eumelanin and phaeomelanin (76). Two principal types of melanin are found in the fungal kingdom. The majority are 1.8-dihydroxynapthalene (DNH) melanins synthesized from acetyl-coenzyme A (CoA) via the polyketide pathway (5). DNH melanins have been found in a wide range of opportunistic fungal pathogens of humans, including dark (dematiaceous) molds, such as Cladosporium, Fonsecaea, Phialophora, and Wangiella species, and as conidial pigments in Aspergillus fumigatus and Aspergillus niger (41, 80, 87, 88). However, several other fungal pathogens, including Blastomyces dermatitidis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii, produce eumelanin (3,4-dihydroxyphenylalanine [DOPA]-melanin) through the activity of a polyphenol oxidase (laccase) and require an exogenous o-diphenolic or p-diphenolic substrate, such as l-DOPA (16, 30, 63,–65, 67, 79).The production of melanin in humans and other mammals is a function of specialized cells called melanocytes. Particles of melanin polymers, sometimes, including more than one melanin type, are built up within membrane-bound organelles called melanosomes (76), and these are actively transported along microtubules to the tips of dendritic outgrowths of melanocytes, from where they are transferred to neighboring cells (32, 81). The mechanism of intercellular transfer of melanosomes has not yet been established, but the export process probably involves the fusion of cell and vesicular membranes rather than secretion of naked melanin (82). In pathogenic fungi, melanins are often reported to be associated with or “in” the cell wall (35, 36, 50, 72, 79). However, there is variation between species: the melanin may be located external to the wall, e.g., in P. brasiliensis (79); within the wall itself (reviewed in reference 42); or as a layer internal to the wall and external to the cell membrane, e.g., in C. neoformans (22, 45, 85). However, mutants of C. neoformans bearing disruptions of three CDA genes involved in the biosynthesis of cell wall chitosan, or of CHS3, encoding a chitin synthase, or of CSR2, which probably regulates Chs3, all released melanin into the culture supernatant, suggesting a role for chitin or chitosan in retaining the pigment polymer in its normal intracellular location (3, 4). However, vesicles externalized from C. neoformans cells also show laccase activity (21), so the effect of chitin may be on vesicle externalization rather than on melanin itself. Internal structures compatible with mammalian melanosomes have been observed in Cladosporium carrionii (73) and in Fonsecaea pedrosoi (2, 26). Remarkably, F. pedrosoi also secretes melanin and locates the polymer within the cell wall (1, 2, 25, 27, 74).Melanization has been found to play an important role in the virulence of several human fungal pathogens, such as C. neoformans, A. fumigatus, P. brasiliensis, S. schenckii, H. capsulatum, B. dermatitidis, and C. posadasii (among recent reviews are references 29, 42, 62, 74, and 79). From these and earlier reviews of the extensive literature, melanin has been postulated to be involved in a range of virulence-associated properties, including interactions with host cells; protection against oxidative stresses, UV light, and hydrolytic enzymes; resistance to antifungal agents; iron-binding activities; and even the harnessing of ionizing radiation in contaminated soils (15). The most extensively studied fungal pathogen for the role of melanization is C. neoformans, which possesses two genes, LAC1 and LAC2, encoding melanin-synthesizing laccases (52, 69, 90). It has been known since early studies with naturally occurring albino variants of C. neoformans (39) that melanin-deficient strains are attenuated in mouse models of cryptococcosis. Deletion of both the LAC1 and LAC2 genes reduced survival of C. neoformans in macrophages (52), and a study based on otherwise isogenic LAC1⁺ and LAC1⁻ strains confirmed the importance of LAC1 in experimental virulence (66). Other genes in the regulatory pathway for LAC1 are similarly known to be essential to virulence (12, 84).C. albicans has been shown to produce melanin with DOPA as a substrate for production of the polymer (53). The cells could be treated with hot acids to produce typical melanin “ghosts,” and antibodies specific for melanin reacted with the fungal cells by immunohistochemistry with tissues from experimentally infected mice, demonstrating that C. albicans produces melanin in vivo (53). However, no candidate genes encoding laccases have yet been identified in the C. albicans genome (http://www.candidagenome.org/). In this study, we investigated the production of melanin by C. albicans and showed that its normal externalization from wild-type cells, including formation of melanosomes, can be altered to an intracellular and intrawall location by mutation of genes involved in chitin synthesis. C. albicans has four genes encoding chitin synthase enzymes. CHS1 is an essential gene under normal conditions (59), and its product is the main enzyme involved in septum formation (83). Chs3 forms the bulk of the chitin in the cell wall and the chitinous ring at sites of bud emergence (8, 51, 57), while Chs2 contributes to differential chitin levels found between yeast and hyphal forms of the fungus, and Chs8 influences the architecture of chitin microfibrils (43, 51, 55, 57, 58). We found that melanin externalization was unaffected in a chs8Δ mutant but was reduced or abrogated in chs2Δ and chs3Δ mutants. Expression profiles of melanin-producing cells grown in the presence of l-DOPA did not identify any potential laccase-synthesizing genes.     

DeMix Workflow for Efficient Identification of Cofragmented Peptides in High Resolution Data-dependent Tandem Mass Spectrometry

Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of “cloning” chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant “one MS/MS spectrum - one peptide” paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs.Shotgun proteomics analysis based on a combination of high performance liquid chromatography and tandem mass spectrometry (MS/MS) (1) has achieved remarkable speed and efficiency (2–7). In a single four-hour long high performance liquid chromatography-MS/MS run, over 40,000 peptides and 5000 proteins can be identified using a high-resolution Orbitrap mass spectrometer with data-dependent acquisition (DDA)¹ (2, 3). However, in a typical LC-MS analysis of unfractionated human cell lysate, over 100,000 individual peptide isotopic patterns can be detected (4), which corresponds to simultaneous elution of hundreds of peptides. With this complexity, a mass spectrometer needs to achieve ≥25 Hz MS/MS acquisition rate to fully sample all the detectable peptides, and ≥17 Hz to cover reasonably abundant ones (4). Although this acquisition rate is reachable by modern time-of-flight (TOF) instruments, the reported DDA identification results do not encompass all expected peptides. Recently, the next-generation Orbitrap instrument, working at 20 Hz MS/MS acquisition rate, demonstrated nearly full profiling of yeast proteome using an 80 min gradient, which opened the way for comprehensive analysis of human proteome in a time efficient manner (5).During the high performance liquid chromatography-MS/MS DDA analysis of complex samples, high density of co-eluting peptides results in a high probability for two or more peptides to overlap within an MS/MS isolation window. With the commonly used ±1.0–2.0 Th isolation windows, most MS/MS spectra are chimeric (4, 8–10), with cofragmenting precursors being naturally multiplexed. However, as has been discussed previously (9, 10), the cofragmentation events are currently ignored in most of the conventional analysis workflows. According to the prevailing assumption of “one MS/MS spectrum–one peptide,” chimeric MS/MS spectra are generally unwelcome in DDA, because the product ions from different precursors may interfere with the assignment of MS/MS fragment identities, increasing the rate of false discoveries in database search (8, 9). In some studies, the precursor isolation width was set as narrow as ±0.35 Th to prevent unwanted ions from being coselected, fragmented or detected (4, 5).On the contrary, multiplexing by cofragmentation is considered to be one of the solid advantages in data-independent acquisition (DIA) (10–13). In several commonly used DIA methods, the precursor ion selection windows are set much wider than in DDA: from 25 Th as in SWATH (12), to extremely broad range as in AIF (13). In order to use the benefit of MS/MS multiplexing in DDA, several approaches have been proposed to deconvolute chimeric MS/MS spectra. In “alternative peptide identification” method implemented in Percolator (14), a machine learning algorithm reranks and rescores peptide-spectrum matches (PSMs) obtained from one or more MS/MS search engines. But the deconvolution in Percolator is limited to cofragmented peptides with masses differing from the target peptide by the tolerance of the database search, which can be as narrow as a few ppm. The “active demultiplexing” method proposed by Ledvina et al. (15) actively separates MS/MS data from several precursors using masses of complementary fragments. However, higher-energy collisional dissociation often produces MS/MS spectra with too few complementary pairs for reliable peptide identification. The “MixDB” method introduces a sophisticated new search engine, also with a machine learning algorithm (9). And the “second peptide identification” method implemented in Andromeda/MaxQuant workflow (16) submits the same dataset to the search engine several times based on the list of chromatographic peptide features, subtracting assigned MS/MS peaks after each identification round. This approach is similar to the ProbIDTree search engine that also performed iterative identification while removing assigned peaks after each round of identification (17).One important factor for spectral deconvolution that has not been fully utilized in most conventional workflows is the excellent mass accuracy achievable with modern high-resolution mass spectrometry (18). An Orbitrap Fourier-transform mass spectrometer can provide mass accuracy in the range of hundreds of ppb (parts per billion) for mass peaks with high signal-to-noise (S/N) ratio (19). However, the mass error of peaks with lower S/N ratios can be significantly higher and exceed 1 ppm. Despite this dependence of the mass accuracy from the S/N level, most MS and MS/MS search engines only allow users to set hard cut-off values for the mass error tolerances. Moreover, some search engines do not provide the option of choosing a relative error tolerance for MS/MS fragments. Such negligent treatment of mass accuracy reduces the analytical power of high accuracy experiments (18).Identification results coming from different MS/MS search engines are sometimes not consistent because of different statistical assumptions used in scoring PSMs. Introduction of tools integrating the results of different search engines (14, 20, 21) makes the data interpretation even more complex and opaque for the user. The opposite trend—simplification of MS/MS data interpretation—is therefore a welcome development. For example, an extremely straightforward algorithm recently proposed by Wenger et al. (22) demonstrated a surprisingly high performance in peptide identification, even though it is only marginally more complex than simply counting the number of matches of theoretical fragment peaks in high resolution MS/MS, without any a priori statistical assumption.In order to take advantage of natural multiplexing of MS/MS spectra in DDA, as well as properly utilize high accuracy of Orbitrap-based mass spectrometry, we developed a simple and robust data analysis workflow DeMix. It is presented in Fig. 1 as an expansion of the conventional workflow. Principles of some of the processes used by the workflow are borrowed from other approaches, including the custom-made mass peak centroiding (20), chromatographic feature detection (19, 20), and two-pass database search with the first limited pass to provide a “software lock mass” for mass scale recalibration (23).Open in a separate windowFig. 1.An overview of the DeMix workflow that expands the conventional workflow, shown by the dashed line. Processes are colored in purple for TOPP, red for search engine (Morpheus/Mascot/MS-GF+), and blue for in-house programs.In DeMix workflow, the deconvolution of chimeric MS/MS spectra consists of simply “cloning” an MS/MS spectrum if a potential cofragmented peptide is detected. The list of candidate peptide precursors is generated from chromatographic feature detection, as in the MaxQuant/Andromeda workflow (16, 19), but using The OpenMS Proteomics Pipeline (TOPP) (20, 24). During the cloning, the precursor is replaced by the new candidate, but no changes in the MS/MS fragment list are made, and therefore the cloned MS/MS spectra remain chimeric. Processing such spectra requires a search engine tolerant to the presence of unassigned peaks, as such peaks are always expected when multiple precursors cofragment. Thus, we chose Morpheus (22) as a search engine. Based on the original search algorithm, we implement a reformed scoring scheme: Morpheus-AS (advanced scoring). It inherits all the basic principles from Morpheus but deeper utilizes the high mass accuracy of the data. This kind of database search removes the necessity of spectral processing for physical separation of MS/MS data into multiple subspectra (15), or consecutive subtraction of peaks (16, 17).Despite the fact that DeMix workflow is largely a combination of known approaches, it provides remarkable improvement compared with the state-of-the-art. On our Orbitrap Q-Exactive workbench, testing on a benchmark dataset of two-hour single-dimension LC-MS/MS experiments from HeLa cell lysate, we identified on average 1.24 peptide per MS/MS spectrum, breaking the “one MS/MS spectrum–one peptide” paradigm on the level of whole data set. At 1% false discovery rate (FDR), we obtained on average nine PSMs per second (at the actual acquisition rate of ca. seven MS/MS spectra per second), and detected 40 human proteins per minute.     

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.Induced pluripotent stem cells (iPSCs)¹ are genetically manufactured pluripotent cells obtained by the transfection of reprogramming factors. Such iPSCs were first reported in 2006 for the mouse (1) and in 2007 for humans (2, 3). Although iPSCs have already been used in the fields of drug development and disease models (4–7), basic aspects of iPSCs largely remain to be elucidated to provide us with a fuller understanding of their properties and for therapeutic applications to be developed in the field of regenerative medicine. These aspects include the need for a definitive system to be established to evaluate their properties; e.g. pluripotency, differentiation propensity, risk of possible contamination of xenoantigens, and even the potential for tumorigenesis. Cell surface glycans are often referred to as the “cell signature,” which changes dramatically depending on the cell properties and conditions (8) as a result of changes in gene expression, including epigenetic modifications of glycan-related molecules. Glycans, because of their outermost cell-surface locations and structural complexity, are considered to be most advantageous communication molecules, playing roles in various biological phenomena. Indeed, SSEA3/4 and Tra-1–60/81, which have been used to discriminate pluripotency, are cell surface glycan epitopes that respond to some specific antibodies (9–12).Glycan-mediated cell-to-cell interactions have been shown to play important roles in various biological phenomena including embryogenesis and carcinogenesis (13–16). This might also be the case for the acquisition and maintenance of iPSC and ESC pluripotency, although there remains much to clarify concerning the roles of cell surface glycans in these events. Thus, the development of novel cell surface markers to evaluate the properties of iPSCs and ESCs is keenly required. Toward this goal, a glycomic approach has been made by several groups (17–20). In our previous study using an advanced lectin microarray technique (21), thirty-eight lectins capable of discriminating between iPSCs and SCs were statistically selected, and the characteristic features of the pluripotent state were obtained. The glycan profiles of the parent SCs, derived from four different tissues, were totally different from one another and from those of the iPSCs. Despite this observation, the technique used lacks the ability to determine detailed glycan structures or allow their quantification. For this purpose, a conventional approach based on high performance liquid chromatography (HPLC) combined with matrix-assisted laser desorption-ionization (MALDI) - time of flight (TOF) mass spectrometry (MS) was undertaken for both the definitive identification of glycan structures and their quantitative comparison, which remained unclear in the previous analysis (21).We report here structural data on N-linked and O-linked glycans derived from the human iPSC 201B7 cell line (2) and human dermal fibroblasts (SC) representing undifferentiated and differentiated cells, respectively. For quantitative comparison, the glycans were liberated by gas-phase hydrazinolysis from similar numbers of cells (22–25) fluorescently tagged with 2-aminopyridine (2-AP) at their reducing terminus (26, 27), following which the derived pyridylaminated (PA-) glycans were purified by multiple-mode (i.e. anion-exchange, size-fractionation and reverse-phase) HPLC. Their structures were determined and quantified by HPLC mapping assisted with MALDI-TOF-MS and exoglycosidase digestion analyses. This report thus provides the first structural evidence showing the occurrence of a dynamic “glycome shift” upon induction of pluripotency.