tion of the ubiquitin–proteasome system with molecular chaperones. The precise association between protein accumulation and cell death remains in-. Chaperone mediated autophagy (CMA): CMA is pretty unique because of a For a protein to be degraded via this pathway, it must be marked by ubiquitin. Role of the ubiquitin/proteasome system in the removal of misfolded proteins .. Through an association of the chaperone with components of the ubiquitin.
Moreover, some ubiquitination associated enzymes—such as E3 ubiquitin ligase—play an irreplaceable role in the formation of acrosome [ 850 — 53 ]. Similarly, the inactivation of the E3 ubiquitin ligase Cullin 4A Cul4 causes male infertility as a consequence of decreased spermatozoa number, reduced sperm motility, and defective acrosome formation [ 51 ]. Interestingly, ubiquitination seems to be an important mechanism of sperm control also in the epididymis, where spermatozoa acquire the motility.
In fact, ubiquitin is present in human seminal plasma [ 54 ], and defective spermatozoa in both humans and animals become ubiquitinated during epididymal passage to be then degraded by the proteasome [ 55 ]. Where there is ubiquitination, deubiquitination also occurs in a reversible manner.
Two classes of deubiquitinating enzymes surely exert their activity at testicular level: Besides the involvement in gonocyte recruitment and cell cycle progression [ 56 ], a growing body of evidences shows the involvement of DUBs especially during the progression into meiotic phase, the spermiogenesis, and the transit in the epididymis in order to direct the formation of high quality sperm and trigger apoptotic mechanisms that recognize and eliminate defective spermatozoa [ 23 ] see Table 1 for details.
Thus, in the next paragraph, we will discuss the conserved role played by the DNAJ protein, Msj-1 mouse sperm cell-specific DNAJ first homologue, currently known as DnaJB3and the DUB enzyme Ubpy ubiquitin-specific processing protease-ycurrently known as Usp8, during the spermiogenesis in both mammals and nonmammalian vertebrates.
Such events are well known from a morphological point of view, but underlying signals and molecular mechanisms leading to them need to be better characterized. In particular, acrosome formation is a key event that has to be checked in order to produce sperm cells of good quality [ 57 ].
However, anterograde and retrograde trafficking pathways of proacrosomic vesicles are controlled step by step to ensure the right timing for fusion [ 59 ]. Such a coordinated process surely needs the participation of testis-specific modulators. The msj-1 gene was first isolated by a mouse spermatogenic cDNA library [ 65 ].
- CHIP: a link between the chaperone and proteasome systems
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Interestingly, its transcript is specifically expressed in germ cells at haploid stages, as the protein appears in spermatids, especially in the periacrosomal and centriolar region in tight association with the testis-specific Hsp and the deubiquitinating enzyme Ubpy [ 636667 ]. A deeper analysis at ultrastructural level reveals Msj-1 close to cellular membranous-vesicular system [ 68 ].
In particular, at the earlier phase of acrosome formation, the Golgi phase, a scattered Msj-1 immunolabelling marks the cytoplasmic area close to proacrosomic granules and Golgi apparatus.
CHIP: a link between the chaperone and proteasome systems
Then, as acrosome formation proceeds to the cap and acrosome phase, in the anterior part of the spermatids, Msj-1 labelling follows the contour of the developing acrosomic vesicles [ 6668 ]. Such studies of localization have prompted to speculate a possible role of Msj-1 in the regulation of acrosome formation. In order to confirm this hypothesis, wobbler mouse has been used as an experimental model [ 64 ].
This is a natural mutant characterized by motoneuron degeneration and defective spermiogenesis with sperm cells lacking a real acrosome and presenting an imperfect head position [ 69 ]. Both defects are due to a missense mutation LQ affecting the gene that codifies Vps54, a vesicular sorting protein, component of Golgi associated retrograde protein GARP complex [ 70 ] that tethers vesicles from endosome to trans-Golgi network [ 71 ].
In particular, endoplasmic reticulum dilatation and abnormal protein accumulation in degenerating motoneurons [ 72 ] as well as missing of fusion of the proacrosomic vesicles during spermiogenesis [ 69 ] have been described in this mutant.
Interestingly, both Msj-1 mRNA and protein have scantly been discovered in wobbler mouse testis, starting from 20 d.
Currently, the downregulation of Msj-1 expression observed in wobbler mouse seems to be combined to an alteration of testicular metabolism. Interestingly, both neurons and spermatozoa possess specialized vesicular organelles such as the neuronal signaling endosome and sperm acrosome, respectively, and both are polarized cells.
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Thus, msj-1 mRNA has also been detected in the central nervous system, at ventral horn motoneuron levels [ 74 ], the other major site of cellular defect in the wobbler mice. In the cervical spinal motoneuron district, msj-1 expression is significantly downregulated [ 74 ], as already observed in testis [ 64 ], thus suggesting that Msj-1 plays a central role in the defects regarding vesicle trafficking linked to the wobbler mutation.
Interestingly, Msj-1 has also been discovered in a lower vertebrate, the anuran amphibian, Rana esculenta, a seasonal breeder whose testis is progressively populated by germ cells at the same stage that develop in germinal cysts [ 75 ].
An expression analysis conducted during the annual sexual cycle revealed the presence of Msj-1 protein in isolated spermatozoa and in testis with a pattern closely associated with the end of meiosis and the onset of spermatid maturation [ 6476 ], exactly as observed in mice [ 6367 ]. Accordingly, an experiment of quiescence induction that causes the depletion of postmeiotic stages, decreases Msj-1 expression, completely confirming the presence of this protein in haploid germ cells [ 64 ].
What is sure is that the use of animal models phylogenetically distant is an important approach for detecting highly conserved molecules. This is the case of Msj-1 that may have a fundamental role during spermiogenesis, especially during acrosomogenesis, even if other functions related to protein folding and misfolding might be postulated.
Beyond Msj-1 expression in frog and mouse, nowadays blast search reveals the presence of DnaJB3 gene homologue in human ID: HeLa cells were lysed in 25 mm Tris, pH 7.
To the cell extracts, antibodies were added as indicated. Rabbit anti-chicken IgGs were used as unrelated control antibody. The immobilized immunocomplexes were collected by centrifugation, washed four times with buffer A containing 0. The protein G-Sepharose was collected by centrifugation, and ATP-eluted proteins were precipitated from the supernatant fraction by addition of trichloroacetic acid.
EDTA was added to a final concentration of 10 mm, and immunoprecipitations were performed as outlined above. Following labeling for 24 h under standard growth conditions, cells were collected and lysed as described above. The factors that influence this molecular triage decision are not clearly understood.
Difficulty with folding and, therefore, the time spent in a partially folded conformation can influence the pathway chosen. Whether this decision is a stochastic process or a regulated and ordered process is also not clear. The J-domain proteins such as Hsp40 are prototypical cochaperones that interact with Hsc70 and enhance adenosine triphosphatase ATPase activity and folding reactions, and more recently, cochaperones with other functions such as conformational stabilization and nucleotide exchange have been identified.
Recent evidence indicates that another cochaperone, CHIP carboxyl terminus of Hsc70 interacting proteinalso regulates chaperone function in part by regulating the molecular triage decision and determining whether proteins enter the productive folding pathway or the degradation pathway. Characterization of CHIP Proteins containing tetratricopeptide repeat TPR domains are involved in many protein-protein interactions Lamb et al ; in particular, several heat shock protein interaction partners—including Hip, Hop, and the cyclophilins—interact with Hsc70 or Hsp90 through TPR domains Ratajczak et al ; Hohfeld et al ; Demand et al In an attempt to identify additional proteins that might be involved in stress regulation, a human heart complementary deoxyribonucleic acid cDNA library was screened with a fragment of cyclophilin containing its 3 carboxy-terminal TPR domains Ballinger et al CHIP was identified through this screen.