The RecD2 helicase balances RecA activities.

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA-ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth.

Bacillus subtilis RadA/Sms-Mediated Nascent Lagging-Strand Unwinding at Stalled or Reversed Forks Is a Two-Step Process: RadA/Sms Assists RecA Nucleation, and RecA Loads RadA/Sms.

Replication fork rescue requires Bacillus subtilis RecA, its negative (SsbA) and positive (RecO) mediators, and fork-processing (RadA/Sms). To understand how they work to promote fork remodeling, reconstituted branched replication intermediates were used. We show that RadA/Sms (or its variant, RadA/Sms C13A) binds to the 5'-tail of a reversed fork with longer nascent lagging-strand and unwinds it in the 5'→3' direction, but RecA and its mediators limit unwinding. RadA/Sms cannot unwind a reversed fork with a longer nascent leading-strand, or a gapped stalled fork, but RecA interacts with and activates unwinding. Here, the molecular mechanism by which RadA/Sms, in concert with RecA, in a two-step reaction, unwinds the nascent lagging-strand of reversed or stalled forks is unveiled. First, RadA/Sms, as a mediator, contributes to SsbA displacement from the forks and nucleates RecA onto single-stranded DNA. Then, RecA, as a loader, interacts with and recruits RadA/Sms onto the nascent lagging strand of these DNA substrates to unwind them. Within this process, RecA limits RadA/Sms self-assembly to control fork processing, and RadA/Sms prevents RecA from provoking unnecessary recombination.

Nucleoid-associated Rok differentially affects chromosomal transformation on Bacillus subtilis recombination-deficient cells.

Rok, a Bacillus subtilis nucleoid-associated protein (NAP), negatively regulates competence development and silences xenogeneic genes. We show that rok inactivation increases rpoB482 natural intraspecies chromosomal transformation (CT) and plasmid transformation to a different extent. In ΔaddAB, ΔrecO, recF15, ΔrecU, ΔruvAB or rec+ cells intraspecies CT significantly increases, but the ΔrecD2 mutation reduces, and the ΔrecX, ΔradA or ΔdprA mutation further decreases CT in the Δrok context when compared to rok+ cells. These observations support the idea that rok inactivation, by altering the topology of the recipient DNA, differentially affects the integration of homologous DNA in rec-deficient strains, and in minor extent the competent subpopulation size. The impairment of other NAP (Hbsu or LrpC) also increased intra- and interspecies CT (nonself-DNA, ~8% nucleotide sequence divergence) in rec+ cells, but differentially reduced both types of CTs in certain rec-deficient strains. We describe that rok inactivation significantly stimulates intra and interspecies CT but differentially reduces them in transformation-deficient cells, perhaps by altering the nucleoid architecture. We extend the observation to other NAPs (Hbsu, LrpC).

Recombination proteins differently control the acquisition of homeologous DNA during Bacillus subtilis natural chromosomal transformation.

Natural chromosomal transformation (CT) plays a major role in prokaryote evolution, yet factors that govern the integration of DNA from related species remain poorly understood. We show that in naturally competent Bacillus subtilis cells the acquisition of homeologous sequences is governed by sequence divergence (SD). Integration initiates in a minimal efficient processing segment via homology-directed CT, and its frequency decreases log-linearly with increased SD up to 15%. Beyond this and up to 23% SD the interspecies boundaries prevail, the CT frequency marginally decreases, and short (<10-nucleotides) segments are integrated via homology-facilitated micro-homologous integration. Both mechanisms are RecA dependent. We identify the other recombination proteins required for the acquisition of homeologous DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair-by-recombination, did not affect CT. However, dprA, radA, recJ, recX or recD2 inactivation strongly decreased intraspecies and interspecies CT. Interspecies CT was not detected beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecX and ~14% in ΔrecD2 cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 accessory proteins are important for the generation of genetic diversity. Together with RecA, they facilitate gene acquisition from bacteria of related species.

Low cost and sustainable hyaluronic acid production in a manufacturing platform based on Bacillus subtilis 3NA strain.

Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genus Streptococcus grown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large-scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption. Bacillus subtilis 3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed. A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100%. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP-free platform that could be adaptable for production of other biomolecules. KEY POINTS: • A biomolecule production platform based on B. subtilis 3NA strain and a synthetic medium was tested for hyaluronic acid biosynthesis • A fermentation process with the maximum volumetric productivity was designed • A techno-economic analysis forecasts a significant reduction in the manufacturing cost compared to the current methods.

Bacillus subtilis RecA interacts with and loads RadA/Sms to unwind recombination intermediates during natural chromosomal transformation.

During natural transformation Bacillus subtilis RecA, polymerized onto the incoming single-stranded (ss) DNA, catalyses DNA strand invasion resulting in a displacement loop (D-loop) intermediate. A null radA mutation impairs chromosomal transformation, and RadA/Sms unwinds forked DNA in the 5'→3' direction. We show that in the absence of RadA/Sms competent cells require the RecG translocase for natural chromosomal transformation. RadA/Sms tetracysteine motif (C13A and C13R) variants, which fail to interact with RecA, are also deficient in plasmid transformation, but this defect is suppressed by inactivating recA. The RadA/Sms C13A and C13R variants bind ssDNA, and this interaction stimulates their ATPase activity. Wild-type (wt) RadA/Sms interacts with and inhibits the ATPase activity of RecA, but RadA/Sms C13A fails to do it. RadA/Sms and its variants, C13A and C13R, bound to the 5'-tail of a DNA substrate, unwind DNA in the 5'→3' direction. RecA interacts with and loads wt RadA/Sms to promote unwinding of a non-cognate 3'-tailed or 5'-fork DNA substrate, but RadA/Sms C13A or C13R fail to do it. We propose that wt RadA/Sms interaction with RecA is crucial to recruit the former onto D-loop DNA, and both proteins in concert catalyse D-loop extension to favour integration of ssDNA during chromosomal transformation.

Bacillus subtilis DisA regulates RecA-mediated DNA strand exchange.

Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork.

DisA Restrains the Processing and Cleavage of Reversed Replication Forks by the RuvAB-RecU Resolvasome.

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

Viral SPP1 DNA is infectious in naturally competent Bacillus subtilis cells: inter- and intramolecular recombination pathways.

A proteolyzed bacteriophage (phage) might release its DNA into the environment. Here, we define the recombination functions required to resurrect an infective lytic phage from inactive environmental viral DNA in naturally competent Bacillus subtilis cells. Using phage SPP1 DNA, a model that accounts for the obtained data is proposed (i) the DNA uptake apparatus takes up environmental SPP1 DNA, fragments it, and incorporates into the cytosol different linear single-stranded (ss) DNA molecules shorter than genome-length; (ii) the SsbA-DprA mediator loads RecA onto any fragmented linear SPP1 ssDNA, but negative modulators (RecX and RecU) promote a net RecA disassembly from these ssDNAs not homologous to the host genome; (iii) single strand annealing (SSA) proteins, DprA and RecO, anneal the SsbA- or SsbB-coated complementary strands, yielding tailed SPP1 duplex intermediates; (iv) RecA polymerized on these tailed intermediates invades a homologous region in another incomplete molecule, and in concert with RecD2 helicase, reconstitutes a complete linear phage genome with redundant regions at the ends of the molecule; and (v) DprA, RecO or viral G35P SSA, may catalyze the annealing of these terminally redundant regions, alone or with the help of an exonuclease, to produce a circular unit-length duplex viral genome ready to initiate replication.

Bacillus subtilis RarA modulates replication restart.

The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance. We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ssDNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA-dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimulate PriA DNA unwinding. In a reconstituted PriA-dependent DNA replication system, RarA inhibited initiation, but not chain elongation. The RarA effect was not observed in the absence of SsbA, or when the host-encoded preprimosome and the DNA helicase are replaced by proteins from the SPP1 phage with similar function. We propose that RarA assembles at blocked forks to maintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarA might impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.

Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation.

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA-SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA-SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA-SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA-SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction.

Natural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or RecO. We show that sequence divergence prevents Bacillus subtilis chromosomal transformation in a log-linear fashion, but it exerts a minor effect when the divergence is localized at a discrete end. In the nucleotide bound form, RecA shows no apparent preference to initiate recombination at the 3'- or 5'-complementary end of the linear duplex with circular ssDNA, but nucleotide hydrolysis is required when heterology is present at both ends. RecA·dATP initiates pairing of the linear 5' and 3' complementary ends, but only initiation at the 5'-end remains stably paired in the absence of SsbA. Our results suggest that during gene transfer RecA·ATP, in concert with SsbA and DprA or RecO, shows a moderate preference for the 3'-end of the duplex. We show that RecA-mediated recombination initiated at the 3'- or 5'-complementary end might have significant implication on the ecological diversification of bacterial species with natural transformation.

Modulation of Lactobacillus casei bacteriophage A2 lytic/lysogenic cycles by binding of Gp25 to the early lytic mRNA.

The genetic switch of Lactobacillus casei bacteriophage A2 is regulated by the CI protein, which represses the early lytic promoter PR and Cro that abolishes expression from the lysogenic promoter PL . Lysogens contain equivalent cI and cro-gp25 mRNA concentrations, i.e., CI only partially represses P(R), predicting a lytic cycle dominance. However, A2 generates stable lysogens. This may be due to Gp25 binding to the cro-gp25 mRNA between the ribosomal binding site and the cro start codon, which abolishes its translation. Upon lytic cycle induction, CI is partially degraded, cro-gp25 mRNA levels increase, and Cro accumulates, launching viral progeny production. The concomitant concentration increase of Gp25 restricts cro mRNA translation, which, together with the low but detectable levels of CI late during the lytic cycle, promotes reentry of part of the cell population into the lysogenic cycle, thus explaining the low proportion of L. casei lysogens that become lysed (∼ 1%). A2 shares its genetic switch structure with many other Firmicutes phages. The data presented may constitute a model of how these phages make the decision for lysis versus lysogeny.

JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation.

Changes in bird-migration patterns associated with human-induced mortality.

Many bird populations have recently changed their migratory behavior in response to alterations of the environment. We collected data over 16 years on male Great Bustards (Otis tarda), a species showing a partial migratory pattern (sedentary and migratory birds coexisting in the same breeding groups). We conducted population counts and radio tracked 180 individuals to examine differences in survival rates between migratory and sedentary individuals and evaluate possible effects of these differences on the migratory pattern of the population. Overall, 65% of individuals migrated and 35% did not. The average distance between breeding and postbreeding areas of migrant individuals was 89.9 km, and the longest average movement of sedentary males was 3.8 km. Breeding group and migration distance had no effect on survival. However, mortality of migrants was 2.4 to 3.5 times higher than mortality of sedentary birds. For marked males, collision with power lines was the main cause of death from unnatural causes (37.6% of all deaths), and migratory birds died in collisions with power lines more frequently than sedentary birds (21.3% vs 6.3%). The percentage of sedentary individuals increased from 17% in 1997 to 45% in 2012. These results were consistent with data collected from radio-tracked individuals: The proportion of migratory individuals decreased from 86% in 1997-1999 to 44% in 2006-2010. The observed decrease in the migratory tendency was not related to climatic changes (temperatures did not change over the study period) or improvements in habitat quality (dry cereal farmland area decreased in the main study area). Our findings suggest that human-induced mortality during migration may be an important factor shaping the migration patterns of species inhabiting humanized landscapes.

The interaction of ω2 with the RNA polymerase ß' subunit functions as an activation to repression switch.

The ω gene is encoded in broad-host range and low-copy plasmids. It is genetically linked to antibiotic resistance genes of the major human pathogens of phylum Firmicutes. The homodimeric forms of ω (ω2) coordinate the plasmid copy number control, faithful partition (ω2 and δ2) and better-than-random segregation (ζϵ2ζ) systems. The promoter (P) of the ωϵζ operon (Pω) transiently interacts with ω2. Adding δ2 facilitates the formation of stable ω2·Pω complexes. Here we show that limiting ω2 interacts with the N-terminal domain of the ß' subunit of the Bacillus subtilis RNA polymerase (RNAP-σ(A)) vegetative holoenzyme. In this way ω2 recruits RNAP-σ(A) onto Pω DNA. Partial Pω occupancy by ω2 increases the rate at which RNAP-σ(A) complex shifts from its closed (RPC) to open (RPO) form. This shift increases transcription activation. Adding δ2 further increases the rate of Pω transcription initiation, perhaps by stabilizing the ω2·Pω complex. In contrast, full operator occupancy by ω2 facilitates RPC formation, but it blocks RPO isomerization and represses Pω utilization. The stimulation and inhibition of RPO formation is the mechanism whereby ω2 mediates copy number fluctuation and stable plasmid segregation. By this mechanism, ω2 also indirectly influences the acquisition of antibiotic resistance genes.

Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair.

Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg(2+) bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to 'activate' RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ-RecQ (RecS) recombinational repair pathways.

Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases.

Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. In this report, we have characterized one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages--i.e., it has both pac and cos sites--a configuration that has not hitherto been described for any mobile element, phages included--and uses the two different phage-coded TerSs. To our knowledge, this is the first example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.

Molecular Anatomy of ParA-ParA and ParA-ParB Interactions during Plasmid Partitioning.

Firmicutes multidrug resistance inc18 plasmids encode parS sites and two small homodimeric ParA-like (δ2) and ParB-like (ω2) proteins to ensure faithful segregation. Protein ω2 binds to parS DNA, forming a short left-handed helix wrapped around the full parS, and interacts with δ2. Protein δ2 interacts with ω2 and, in the ATP-bound form, binds to nonspecific DNA (nsDNA), forming small clusters. Here, we have mapped the ω2·Î´2 and δ2·Î´2 interacting domains in the δ2 that are adjacent to but distinct from each other. The δ2 nsDNA binding domain is essential for stimulation of ω2·parS-mediated ATP hydrolysis. From the data presented here, we propose that δ2 interacts with ATP, nsDNA, and with ω2 bound to parS at near equimolar concentrations, facilitating a δ2 structural transition. This δ2 "activated" state overcomes its impediment in ATP hydrolysis, with the subsequent release of both of the proteins from nsDNA (plasmid unpairing).

Hyperosmotic Stress Induces Tau Proteolysis by Caspase-3 Activation in SH-SY5Y Cells.

Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of µ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor µ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. J. Cell. Biochem. 117: 2781-2790, 2016. © 2016 Wiley Periodicals, Inc.