Proton and calcium pumping P-type ATPases and their regulation of plant responses to the environment

Plant plasma membrane H⁺-ATPases and Ca²⁺-ATPases maintain low cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H⁺-ATPases to function as electrogenic voltage stats, and Ca²⁺-ATPases as “off” mechanisms in Ca²⁺-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H⁺ and Ca²⁺, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.

The regulation of plasma membrane H^+-ATPases and autoinhibited Ca2^+-ATPases exhibits a complex and dynamic network of posttranslational regulation. The regulation of plasma membrane H⁺-ATPases and autoinhibited Ca²⁺-ATPases exhibits a complex and dynamic network of posttranslational regulation.

P-type ATPases are found in all domains of life and constitute a large superfamily of membrane-bound pumps that share a common machinery, including a reaction cycle that involves catalytic phosphorylation of an Asp, resulting in a phosphorylated intermediate (reviewed in Palmgren and Nissen, 2011; (hence the name P-type; Box 1). The catalytic phosphoryl-aspartate intermediate is not to be confused with regulatory phosphorylation, which occurs on Ser, Thr, and Tyr residues. Five major families of P-type ATPases have been characterized (P1–5), each of which is divided into a number of subfamilies (named with letters). Plasma membrane H⁺-ATPases are classified as P3A ATPases, whereas Ca²⁺ pumps constitute P2A and P2B ATPases. In plants, these pumps are best characterized in the model plant Arabidopsis thaliana (Arabidopsis).Box 1Enzymology of P-type ATPases.P-type ATPases (reviewed in Palmgren and Nissen, 2011) alternate between two extreme conformations during their catalytic cycle: a high-affinity (with respect to ATP and the ion to be exported) Enzyme1 (E1) state, and a low-affinity Enzyme2 (E2) state. Many P-type ATPases are autoinhibited by built-in molecular constraints, namely their C- and N-terminal (for plasma membrane H⁺-ATPases; Palmgren et al., 1999) or N-terminal (for P2B Ca²⁺-ATPases; Malmström et al., 1997) regulatory (R) domains of approximately 100 amino acid residues, which act as brakes by stabilizing the pumps in a low-affinity conformation (Palmgren and Nissen, 2011), most likely E2. Neutralizing the R domain results in a shift in conformational equilibrium towards a high-affinity state, likely E1. In this way, the R domains of plasma membrane H⁺-ATPases and Ca²⁺-ATPases allow posttranslational modification events to control the turnover numbers of these pumps. A structure of a plasma membrane H⁺-ATPase (from the distantly related yeast S. cerevisiae) in its autoinhibited state has been solved (Heit et al., 2021). Its R domain is situated adjacent to the P domain, which would suggest that the R domain functions to restrict the conformational flexibility of the pump. Normally, the hydrolysis of ATP and transport are tightly coupled in P-type ATPases. Therefore, P-type ATPases hydrolyze bound ATP as soon as their ligand-binding site(s) in the membrane region are occupied, but not before. Thus, increasing the ligand affinity of an ATPase simultaneously increases its turnover number, provided that the concentration of ATP is not limiting, which is rarely the case in cells. A specific feature of plasma membrane H⁺-ATPases is that in the autoinhibited state, ATP hydrolysis is only loosely coupled to H⁺ pumping, whereas pump activation results in tight coupling, with one H⁺ pumped per ATP split (Pedersen et al., 2018).In response to internal and/or external cues, plasma membrane H⁺-ATPase and Ca²⁺-ATPase activities are controlled by intracellular concentrations of H⁺ and Ca²⁺, respectively, via interacting proteins, through posttranslational modification by phosphorylation, and by regulated trafficking of the pump to and from the plasma membrane. Their regulation sometimes involves changes in gene expression and turnover, although this is rare, perhaps because both processes are time- and energy-consuming (Haruta et al., 2018).     

Defective Satellite DNA Clustering into Chromocenters Underlies Hybrid Incompatibility in Drosophila

Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI) (Yunis and Yasmineh 1971; Henikoff et al. 2001; Ferree and Barbash 2009; Sawamura 2012; Jagannathan and Yamashita 2017), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into “chromocenters,” thereby bundling chromosomes to maintain the entire genome in a single nucleus (Jagannathan et al. 2018, 2019). Here, we show that ineffective clustering of divergent satellite DNA in the cells of Drosophila hybrids results in chromocenter disruption, associated micronuclei formation, and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation.     

Salt Stress Reduces Root Meristem Size by Nitric Oxide-Mediated Modulation of Auxin Accumulation and Signaling in Arabidopsis

The development of the plant root system is highly plastic, which allows the plant to adapt to various environmental stresses. Salt stress inhibits root elongation by reducing the size of the root meristem. However, the mechanism underlying this process remains unclear. In this study, we explored whether and how auxin and nitric oxide (NO) are involved in salt-mediated inhibition of root meristem growth in Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic approaches. We found that salt stress significantly reduced root meristem size by down-regulating the expression of PINFORMED (PIN) genes, thereby reducing auxin levels. In addition, salt stress promoted AUXIN RESISTANT3 (AXR3)/INDOLE-3-ACETIC ACID17 (IAA17) stabilization, which repressed auxin signaling during this process. Furthermore, salt stress stimulated NO accumulation, whereas blocking NO production with the inhibitor N^ω-nitro-l-arginine-methylester compromised the salt-mediated reduction of root meristem size, PIN down-regulation, and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated inhibition of root meristem growth. Taken together, these findings suggest that salt stress inhibits root meristem growth by repressing PIN expression (thereby reducing auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via increasing NO levels.Due to agricultural practices and climate change, soil salinity has become a serious factor limiting the productivity and quality of agricultural crops (Zhu, 2007). Worldwide, high salinity in the soil damages approximately 20% of total irrigated lands and takes 1.5 million ha out of production each year (Munns and Tester, 2008). In general, high salinity affects plant growth and development by reducing plant water potential, altering nutrient uptake, and increasing the accumulation of toxic ions (Hasegawa et al., 2000; Munns, 2002; Zhang and Shi, 2013). Together, these effects severely reduce plant growth and survival.Because the root is the first organ to sense high salinity, salt stress plays a direct, important role in modulating root system architecture (Wang et al., 2009). For instance, salt stress negatively regulates root hair formation and gravitropism (Sun et al., 2008; Wang et al., 2008). The role of salt in lateral root formation depends on the NaCl concentration. While high NaCl levels inhibit lateral root formation, lower NaCl levels stimulate lateral root formation in an auxin-dependent manner (Zolla et al., 2010; Ji et al., 2013). The root meristem plays an essential role in sustaining root growth (Perilli et al., 2012). Salt stress inhibits primary root elongation by suppressing root meristem activity (West et al., 2004). However, how this inhibition occurs remains largely unclear.Plant hormones are important intermediary signaling compounds that function downstream of environmental stimuli. Among plant hormones, indole-3-acetic acid (IAA) is thought to play a fundamental role in root system architecture by regulating cell division, expansion, and differentiation. In Arabidopsis (Arabidopsis thaliana) root tips, a distal auxin maximum is formed and maintained by polar auxin transport (PAT), which determines the orientation and extent of cell division in the root meristem as well as root pattern formation (Sabatini et al., 1999). PINFORMED (PIN) proteins, which are components of the auxin efflux machinery, regulate primary root elongation and root meristem size (Blilou et al., 2005; Dello Ioio et al., 2008; Yuan et al., 2013, 2014). The auxin signal transduction pathway is activated by direct binding of auxin to its receptor protein, TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB), promoting the degradation of Aux/IAA proteins, releasing auxin response factors (ARFs), and activating the expression of auxin-responsive genes (Gray et al., 2001; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). Aux/IAA proteins are short-lived, nuclear-localized proteins that play key roles in auxin signal activation and root growth modulation (Rouse et al., 1998). Other hormones and stresses often regulate auxin signaling by affecting Aux/IAA protein stability (Lim and Kunkel, 2004; Nemhauser et al., 2004; Wang et al., 2007; Kushwah and Laxmi, 2014).Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants (He et al., 2004; Fernández-Marcos et al., 2011; Shi et al., 2012), including important roles in the regulation of root growth and development. NO functions downstream of auxin during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002). Exogenous auxin-induced NO biosynthesis is associated with nitrate reductase activity during lateral root formation, and NO is necessary for auxin-induced lateral root and root hair development (Pagnussat et al., 2002; Lombardo et al., 2006). Pharmacological and genetic analyses in Arabidopsis indicate that NO suppresses primary root growth and root meristem activity (Fernández-Marcos et al., 2011). Additionally, both exogenous application of the NO donor sodium nitroprusside (SNP) and overaccumulation of NO in the mutant chlorophyll a/b binding protein underexpressed1 (cue1)/nitric oxide overproducer1 (nox1) result in reduced PIN1 expression and auxin accumulation in root tips. The auxin receptors protein TIR1 is S-nitrosylated by NO, suggesting that this protein is a direct target of NO in the regulation of root development (Terrile et al., 2012).Because NO is a free radical, NO levels are dynamically regulated by endogenous and environmental cues. Many phytohormones, including abscisic acid, auxin, cytokinin, salicylic acid, jasmonic acid, and ethylene, induce NO biosynthesis (Zottini et al., 2007; Kolbert et al., 2008; Tun et al., 2008; García et al., 2011). In addition, many abiotic and biotic stresses or stimuli, such as cold, heat, salt, drought, heavy metals, and pathogens/elicitors, also stimulate NO biosynthesis (Zhao et al., 2009; Mandal et al., 2012). Salt stress stimulates NO and ONOO^– accumulation in roots (Corpas et al., 2009), but the contribution of NO to root meristem growth under salinity stress has yet to be examined in detail.In this study, we found that salt stress significantly down-regulated the expression of PIN genes and promoted AUXIN RESISTANT3 (AXR3)/IAA17 stabilization. Furthermore, salt stress stimulated NO accumulation, and pharmacological inhibition of NO biosynthesis compromised the salt-mediated reduction in root meristem size. Our results support a model in which salt stress reduces root meristem size by increasing NO accumulation, which represses PIN expression and stabilizes IAA17, thereby reducing auxin levels and repressing auxin signaling.     

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.     

Phytochrome B Promotes Branching in Arabidopsis by Suppressing Auxin Signaling

Many plants respond to competition signals generated by neighbors by evoking the shade avoidance syndrome, including increased main stem elongation and reduced branching. Vegetation-induced reduction in the red light:far-red light ratio provides a competition signal sensed by phytochromes. Plants deficient in phytochrome B (phyB) exhibit a constitutive shade avoidance syndrome including reduced branching. Because auxin in the polar auxin transport stream (PATS) inhibits axillary bud outgrowth, its role in regulating the phyB branching phenotype was tested. Removing the main shoot PATS auxin source by decapitation or chemically inhibiting the PATS strongly stimulated branching in Arabidopsis (Arabidopsis thaliana) deficient in phyB, but had a modest effect in the wild type. Whereas indole-3-acetic acid (IAA) levels were elevated in young phyB seedlings, there was less IAA in mature stems compared with the wild type. A split plate assay of bud outgrowth kinetics indicated that low auxin levels inhibited phyB buds more than the wild type. Because the auxin response could be a result of either the auxin signaling status or the bud’s ability to export auxin into the main shoot PATS, both parameters were assessed. Main shoots of phyB had less absolute auxin transport capacity compared with the wild type, but equal or greater capacity when based on the relative amounts of native IAA in the stems. Thus, auxin transport capacity was unlikely to restrict branching. Both shoots of young phyB seedlings and mature stem segments showed elevated expression of auxin-responsive genes and expression was further increased by auxin treatment, suggesting that phyB suppresses auxin signaling to promote branching.The development of shoot branches is a multistep process with many potential points of regulation. After the formation of an axillary meristem in the leaf axil, an axillary bud may form through the generation of leaves and other tissues. The axillary bud may grow out to form a branch, or may remain dormant or semidormant for an indefinite period of time (Bennett and Leyser, 2006). In Arabidopsis (Arabidopsis thaliana), the position of the bud in the rosette is a strong determinant of its fate, with upper buds displaying greater outgrowth potential than lower buds. In fact, the varying potential of buds at different positions is maintained even in buds that are activated to form branches, with the upper buds growing out first and most robustly, and lower buds growing out after a time lag and with less vigor (Hempel and Feldman, 1994; Finlayson et al., 2010).The disparate fate of buds at different rosette positions is mediated, at least in part, by the process of correlative inhibition, whereby remote parts of the plant inhibit the outgrowth of the buds (Cline, 1997). Correlative inhibition is typically associated with the bud-inhibiting effects of auxin sourced in the shoot apex and transported basipetally in the polar auxin transport stream (PATS). Auxin in the PATS does not enter the bud and thus must act indirectly; however, the exact mechanism by which auxin inhibits bud outgrowth is not well understood, despite many years of intensive study (Waldie et al., 2010; Domagalska and Leyser, 2011). Evidence supports divergent models by which auxin may regulate branching. One model contends that the PATS modulates a bud outgrowth inhibiting second messenger (Brewer et al., 2009). Another model postulates a mechanism whereby competition between the main shoot and the axillary bud for auxin export in the PATS regulates bud activity (Bennett et al., 2006; Prusinkiewicz et al., 2009; Balla et al., 2011).In addition to intrinsic developmental programming, branching is also modulated by environmental signals, including competition signals generated by neighboring plants. The red light:far-red light ratio (R:FR) is an established competition signal that is modified (reduced) by neighboring plants and sensed by the phytochrome family of photoreceptors. A low R:FR evokes the shade avoidance syndrome with plants displaying, among other phenotypes, enhanced shoot elongation and reduced branching (Smith, 1995; Ballaré, 1999; Franklin and Whitelam, 2005; Casal, 2012). Phytochrome B (phyB) is the major sensor contributing to R:FR responses, and loss of phyB function results in a plant that displays a phenotype similar to constitutive shade avoidance. It should be noted that actual shade avoidance is mediated by additional phytochromes and that the complete absence of functional phyB in the loss-of-function mutant may also result in a phenotype that does not exactly mirror shade avoidance. Loss of phyB function leads to reduced branching and altered expression of genes associated with hormone pathways and bud development in the axillary buds (Kebrom et al., 2006; Finlayson et al., 2010; Kebrom et al., 2010; Su et al., 2011). In Arabidopsis, phyB deficiency differentially affects the outgrowth of buds from specific positions in the rosette and thus demonstrates an important function in the regulation of correlative inhibition (Finlayson et al., 2010; Su et al., 2011), a process known to be influenced by auxin. Many aspects of auxin signaling are dependent on AUXIN RESISTANT1 (AXR1), which participates in activating the Skip-Cullin-F-box auxin signaling module (del Pozo et al., 2002). Reduced auxin signaling resulting from AXR1 deficiency enabled phyB-deficient plants to branch profusely and reduced correlative inhibition, thus establishing auxin signaling downstream of phyB action (Finlayson et al., 2010). Although a link between auxin signaling and phyB regulation of branching was demonstrated, the details of the interaction were not discovered.The relationship between auxin and shade avoidance responses has been investigated in some detail. Auxin signaling was implicated in shade avoidance responses mediated by ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 in young Arabidopsis seedlings (Steindler et al., 1999). Rapid changes in leaf development resulting from canopy shade were also shown to involve TRANSPORT INHIBITOR RESPONSE1-dependent auxin signaling (Carabelli et al., 2007). A link between auxin abundance and the response to the R:FR was demonstrated in Arabidopsis deficient for the TRP AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) auxin biosynthetic enzyme (Tao et al., 2008). Young wild-type seedlings respond to a decreased R:FR by increasing indole-3-acetic acid (IAA) biosynthesis, accumulating IAA, increasing hypocotyl and petiole elongation, and increasing leaf elevation. However, these responses are reduced in plants deficient in TAA1. Subsequent studies confirmed the importance of auxin in responses to the R:FR (Pierik et al., 2009; Kozuka et al., 2010; Keller et al., 2011), and also identified the auxin transporter PIN-FORMED3 as essential for hypocotyl elongation responses in young seedlings (Keuskamp et al., 2010). In addition to the roles of auxin abundance and transport in the process, auxin sensitivity has also been implicated in shade avoidance. Several auxin signaling genes are direct targets of the phytochrome signaling component PHYTOCHROME INTERACTING FACTOR5 (PIF5), and these genes are misregulated in Arabidopsis deficient in either PHYTOCHROME INTERACTING FACTOR4 (PIF4) or PIF5 (Hornitschek et al., 2012; Sun et al., 2013). Auxin-responsive hypocotyl elongation and auxin-induced gene expression were also reduced in young seedlings of the pif4pif5 double mutant (Hornitschek et al., 2012), which show defects in shade avoidance responses (Lorrain et al., 2008).Although some aspects of the regulation of branching are now understood, there are still many gaps in our knowledge of the process, especially as related to the regulation of branching by light signals. Because auxin is known to play a major role in regulating branch development, and because recent studies have implicated auxin in general shade avoidance responses and specifically in the regulation of branching by phyB, the hypothesis that auxin homeostasis, transport, and/or signaling may contribute to the hypobranching phenotype of phyB-deficient plants was generated and tested, using a variety of physiological and molecular approaches.     

MULTI-FLORET SPIKELET1, Which Encodes an AP2/ERF Protein,Determines Spikelet Meristem Fate and Sterile Lemma Identity in Rice

The spikelet is a unique inflorescence structure of grass. The molecular mechanism that controls the development of the spikelet remains unclear. In this study, we identified a rice (Oryza sativa) spikelet mutant, multi-floret spikelet1 (mfs1), that showed delayed transformation of spikelet meristems to floral meristems, which resulted in an extra hull-like organ and an elongated rachilla. In addition, the sterile lemma was homeotically converted to the rudimentary glume and the body of the palea was degenerated in mfs1. These results suggest that the MULTI-FLORET SPIKELET1 (MFS1) gene plays an important role in the regulation of spikelet meristem determinacy and floral organ identity. MFS1 belongs to an unknown function clade in the APETALA2/ethylene-responsive factor (AP2/ERF) family. The MFS1-green fluorescent protein fusion protein is localized in the nucleus. MFS1 messenger RNA is expressed in various tissues, especially in the spikelet and floral meristems. Furthermore, our findings suggest that MFS1 positively regulates the expression of LONG STERILE LEMMA and the INDETERMINATE SPIKELET1 (IDS1)-like genes SUPERNUMERARY BRACT and OsIDS1.In the reproductive phase of angiosperms, the shoot meristem is transformed into an inflorescence meristem, which then produces a floral meristem from which floral organs begin to develop, according to the mechanism known as the ABCDE model (Coen and Meyerowitz, 1991; Coen and Nugent, 1994; Dreni et al., 2007; Ohmori et al., 2009). An inflorescence can be classified as determinate or indeterminate based on whether its apical meristem is transformed into a terminal floral meristem. In an indeterminate inflorescence, the lateral meristem is permanently differentiated from the apical meristem, which is not converted into the terminal floral meristem, as occurs during the development of the inflorescences of Arabidopsis (Arabidopsis thaliana) and snapdragon (Antirrhinum majus). In contrast, in a determinate inflorescence, the apical meristem is transformed into the terminal floral meristem after the production of a fixed number of lateral meristems, as occurs during the development of the inflorescences of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum; Bradley et al., 1997; Ratcliffe et al., 1999; Sussex and Kerk, 2001; Chuck et al., 2008).In general, inflorescences in grasses consist of branches and spikelets (Coen and Nugent, 1994; Itoh et al., 2005; Kobayashi et al., 2010). In these organisms, the branch meristem is determinate. It produces several lateral spikelet meristems, followed by the final production of a terminal spikelet meristem. The spikelet, the specific unit of the grass inflorescence, comprises a pair of bracts and one to 40 florets; it shows determinacy or indeterminacy depending on the species (Clifford, 1987; Malcomber et al., 2006). In species with a determinate spikelet, such as rice (Oryza sativa), after the production of fixed lateral floral meristems, the spikelet meristems are converted into terminal floral meristems, resulting in termination of the spikelet meristem fate. In contrast, in species with an indeterminate spikelet, such as wheat (Triticum aestivum), the spikelet meristem fate is maintained continuously and produces a variable number of lateral floral meristems.In Arabidopsis, the gene TERMINAL FLOWER1 (TFL1) was shown to maintain indeterminacy in the fate of the inflorescence. In the tfl1 mutant, the inflorescence meristems were converted into floral meristems earlier than in the wild type, but the ectopic expression of TFL1 resulted in the transformation of floral meristems at a later stage of development to secondary inflorescence meristems (Bradley et al., 1997; Ratcliffe et al., 1999; Mimida et al., 2001). In rice, overexpression of either of the TFL1-like genes, RICE CENTRORADIALIS1 (RCN1) or RCN2, delayed the transition of branch meristems to spikelet meristems and finally resulted in the production of a greater number of branches and spikelets than in the wild type (Nakagawa et al., 2002; Rao et al., 2008).To date, no gene that acts to maintain the indeterminacy of the spikelet meristem has been reported. However, two classes of genes have been shown to be involved in termination of the indeterminacy of spikelet meristems. One of these is the group of terminal floral meristem identity genes. A grass-specific LEAFY HULL STERILE1 (LHS1) clade in the SEPALLATA (SEP) subfamily belongs to this class. LHS1-like genes were found to be expressed only in the terminal floral meristem in species with spikelet determinacy, which suggested that they exclusively determine the production of the terminal floral meristem, by which the spikelet meristem acquires determinacy (Cacharroón et al., 1999; Malcomber and Kellogg, 2004; Zahn et al., 2005). The other class comprises the INDETERMINATE SPIKELET1 (IDS1)-like genes, which belong to the APETALA2/ethylene-responsive factor (AP2/ERF) family. Unlike LHS1-like genes, this class of genes regulates the correct timing of the transition of the spikelet meristem to the floral meristem but does not specify the identity of the terminal floral meristem. In maize (Zea mays), loss of IDS1 function produces extra florets (Chuck et al., 1998). In addition, mutation of SISTER OF IDS1 (SID1), a paralog of IDS1 in maize, resulted in no defects in terms of spikelet development. However, the ids1+sid1 double mutant failed to generate floral organs and instead developed more bract-like structures than are found in wild-type plants (Chuck et al., 2008). The rice genome contains two IDS1-like genes, SUPERNUMERARY BRACT (SNB) and OsIDS1. Loss of activity of SNB or OsIDS1 produced extra rudimentary glumes, and snb+osids1 double mutant plants developed more rudimentary glumes than either of its parental mutants (Lee et al., 2007; Lee and An, 2012). These results revealed that the mutated IDS1-like genes prolonged the activity of the spikelet meristem.In most members of Oryzeae, the spikelet is distinct from those of other grasses, in that it comprises a pair of rudimentary glumes, a pair of sterile lemmas (empty glumes), and one floret (Schmidt and Ambrose, 1998; Ambrose et al., 2000; Kellogg, 2009; Hong et al., 2010). The rudimentary glumes are generally regarded as severely reduced bract organs, but the origin of sterile lemmas has been widely debated. Recent studies suggested that the sterile lemmas are the vestigial lemmas of two lateral florets. The gene LONG STERILE LEMMA (G1)/ELONGATED EMPTY GLUME1 (ELE1) is a member of a plant-specific gene family. In the g1/ele1 mutant, sterile lemmas were found to be homeotically transformed into lemmas (Yoshida et al., 2009; Hong et al., 2010). The OsMADS34 and EXTRA GLUME1 (EG1) genes were also shown to determine the identities of sterile lemmas. In the osmads34 and eg1 mutants, the sterile lemmas were enlarged and acquired the identities of lemmas (Li et al., 2009; Gao et al., 2010; Kobayashi et al., 2010). Additionally, the SEP-like gene LHS1/OsMADS1, which specifies the identities of both the lemma and the palea, was not expressed in sterile lemmas, and ectopic expression in sterile lemmas resulted in the transformation of sterile lemmas to lemmas (Jeon et al., 2000; Li et al., 2009; Tanaka et al., 2012). These findings suggest that the sterile lemma may be homologous to the lemma. Nevertheless, some researchers still considered that the sterile lemmas are instead vestigial bract-like structures similar to the rudimentary glumes (Schmidt and Ambrose, 1998; Kellogg, 2009; Hong et al., 2010).In this study, we isolated the rice MULTI-FLORET SPIKELET1 (MFS1) gene, which belongs to a clade of unknown function in the AP2/ERF gene family. The mutation of MFS1 was shown to delay the transformation of the spikelet meristem to the floral meristem and to result in degeneration of the sterile lemma and palea. These results suggest that MFS1 plays an important role in the regulation of spikelet determinacy and organ identity. Our findings also reveal that MFS1 positively regulates the expression of G1 and the IDS1-like genes SNB and OsIDS1.