ArHsp40, a type 1 J-domain protein, is developmentally regulated and stress inducible in post-diapause Artemia franciscana

Abstract

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during postdiapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/ E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.

Post-diapause synthesis of ArHsp40-2, a type 2 J-domain protein from Artemia franciscana, is developmentally regulated and induced by stress

“…ArHsp40, a type 1 J-domain protein from A . franciscana has been described [ 29 ] but no other J-domain proteins are reported for this organism. To approach the question of Hsp40 diversity and function in A .…”

“…As a consequence, a type 2 J-domain protein termed ArHsp40-2, which lacks a zinc binding domain, was found. The synthesis of ArHsp40-2, like ArHsp40 [ 29 ] was heat inducible, indicating a role in stress tolerance. However the synthesis pattern of ArHsp40-2 during post-diapause development was different than that of ArHsp40 and the time of maximum accumulation suggested a role for ArHsp40-2 in the recovery of proteins during post-diapause development.…”

ArHsp40 and ArHsp40-2 contribute to stress tolerance and longevity in Artemia franciscana, but only ArHsp40 influences diapause entry

“…To prepare dsRNA, Invitrogen™ Platinum Taq DNA Polymerase (Fisher Scientific, Ottawa, ON, Canada) and primers (Integrated DNA Technologies, Coralville, IA, USA) containing the T7 promoter (Table 1) were used to amplify ArHsp40-1 (Jiang et al, 2016) and ArHsp40-2 (Rowarth and MacRae, 2018) cDNAs by PCR. Green fluorescent protein (GFP) cDNA was amplified from the vector pEGFP-N1 (Clontech, Mountain View, CA, USA) (King and MacRae, 2012).…”

“….8], placed in a boiling water bath for 5 min and then centrifuged at 4°C for 10 min at 10,000 g. Proteins were resolved in 12.5% SDS polyacrylamide gels, transferred to 0.2 µm nitrocellulose membranes (Bio-Rad, Mississauga, ON, Canada) overnight at 100 mA and blocked for 1 h at room temperature in 5% (w/v) Carnation low-fat milk powder in TBS (10 mmol l −1 Tris, 140 mmol l −1 NaCl, pH 7.4). Membranes were then probed with the antibodies Anti40-type 1 (Jiang et al, 2016), Anti40-type 2 (Rowarth and MacRae, 2018) or Anti-Y (Xiang and MacRae, 1995), each diluted 1:1000 in TBS for 15 min followed by washing in TBS-T (10 mmol l −1 Tris, 140 mmol l −1 NaCl, 0.1% Tween-20, pH 7.4) for 1, 2, 3 and 4 min. Subsequent to washing, membranes were probed for 20 min with HRP-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) diluted 1:10,000 in TBS and washed as before in TBS-T followed by TBS for 3 min.…”

“…Two J-domain proteins have been identified in A. franciscana, namely the type 1 ArHsp40 and the type 2 ArHsp40-2 (Jiang et al, 2016;Rowarth and MacRae, 2018). These J-domain proteins are induced by heat shock in nauplii, indicating a role in stress tolerance, and both are developmentally regulated during post-diapause development of A. franciscana; however, their patterns of synthesis vary, suggesting different functions.…”