Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Mcl-1 Degradation during Hepatocyte Lipoapoptosis

The mechanisms of free fatty acid-induced lipoapoptosis are incompletely understood. Here we demonstrate that Mcl-1, an anti-apoptotic member of the Bcl-2 family, was rapidly degraded in hepatocytes in response to palmitate and stearate by a proteasome-dependent pathway. Overexpression of a ubiquitin-resistant Mcl-1 mutant in Huh-7 cells attenuated palmitate-mediated Mcl-1 loss and lipoapoptosis; conversely, short hairpin RNA-targeted knockdown of Mcl-1 sensitized these cells to lipoapoptosis. Palmitate-induced Mcl-1 degradation was attenuated by the novel protein kinase C (PKC) inhibitor rottlerin. Of the two human novel PKC isozymes, PKCδ and PKCθ, only activation of PKCθ was observed by phospho-immunoblot analysis. As compared with Jurkat cells, a smaller PKCθ polypeptide and mRNA were expressed in hepatocytes consistent with an alternative splice variant. Short hairpin RNA-mediated knockdown of PKCθ reduced Mcl-1 degradation and lipoapoptosis. Likewise, genetic deletion of Pkcθ also attenuated Mcl-1 degradation and cytotoxicity by palmitate in primary hepatocytes. During treatment with palmitate, rottlerin inhibited phosphorylation of Mcl-1 at Ser¹⁵⁹, a phosphorylation site previously implicated in Mcl-1 turnover. Consistent with these results, an Mcl-1 S159A mutant was resistant to degradation and improved cell survival during palmitate treatment. Collectively, these results implicate PKCθ-dependent destabilization of Mcl-1 as a mechanism contributing to hepatocyte lipoapoptosis.Current evidence suggests that hepatic steatosis is present in up to 30% of the American population (1). A subset of these individuals develop severe hepatic lipotoxicity, a syndrome referred to as NASH² (2), which can progress to cirrhosis and its chronic sequela (3, 4). A major risk factor for hepatic lipotoxicity is insulin resistance (5–7), resulting in excessive lipolysis within peripheral adipose tissue with release of high levels of free fatty acids (FFA) to the circulation. Circulating FFA are taken up by the liver via fatty acid transporter 5 and CD36 (8–10), and the bulk of hepatic neutral fat is derived from re-esterification of circulating FFA (8). Current concepts indicate that FFA, and not their esterified product (triglyceride), mediate hepatic lipotoxicity (11, 12). Elevated serum FFA correlate with liver disease severity (13–15), and therapies that enhance insulin sensitivity ameliorate hepatic lipotoxicity, in part, by decreasing plasma FFA (16). Hepatic FFA also accumulate in experimental steatohepatitis, further supporting a role for these nutrients in hepatic lipotoxicity (17). Saturated FFA are more strongly implicated in hepatic lipotoxicity than unsaturated FFA (18, 19). Saturated FFA induce hepatocyte apoptosis (20, 21), a cardinal feature of nonalcoholic fatty liver disease (22), and serum biomarkers of apoptosis are useful for identifying hepatic lipotoxicity (23). Thus, FFA-mediated lipotoxicity occurs, in part, by apoptosis.Apoptosis is regulated by members of the Bcl-2 protein family (24). These proteins can be categorized into three subsets as follows: the guardians or anti-apoptotic members of this family, which include Bcl-2, A1, Mcl-1, Bcl-x_L, and Bcl-w; the multidomain executioners or proapoptotic members of this family, which include Bax and Bak; and the messengers or biosensors of cell death, which share only the third Bcl-2 homology domain and are referred to as BH3-only proteins. This last group of proteins includes Bid, Bim, Bmf, Puma, Noxa, Hrk, Bad, and Bik. We have previously reported that cytotoxic FFA induce Bim expression by a FoxO3a-dependent mechanism that contributes, in part, to lipoapoptosis by activating Bax (20, 21). However, Bax activation can be held in check by anti-apoptotic members of the Bcl-2 family suggesting their function may also be dysregulated during FFA-mediated cytotoxicity.Bcl-2 is not expressed in hepatocytes at the protein level (25), whereas Bcl-w and Bfl-1/A1 knock-out mice have no liver phenotype (26–28). However, both potent anti-apoptotic proteins Bcl-x_L and Mcl-1 are expressed by hepatocytes and exhibit a liver phenotype in knock-out mice (29, 30), whereas up-regulation of Mcl-1 renders hepatocytes resistant to apoptosis (31–33). It has also been posited that cellular elimination of Mcl-1 is a critical step in certain proapoptotic cascades (34, 35). Mcl-1 is unique among Bcl-2 proteins in that it has a short half-life, 30–120 min in most cell types, due to the presence of two sequences rich in proline, glutamic acid, serine, and threonine, which target the protein for rapid degradation by the proteasome (36). Proteasomal degradation of Mcl-1 is promoted by ubiquitination, which in turn is regulated by various kinase cascades (36). Despite its potential importance, a role for Mcl-1 in regulating hepatocyte FFA-mediated lipoapoptosis remains unexplored.Given that FFA induce insulin resistance (37), the kinases potentially regulating lipoapoptosis are likely those also identified in insulin resistance syndromes, especially the novel PKC isoforms PKCδ and PKCθ (38). The novel PKC isoforms are activated by diacylglycerol, which rises in the presence of FFA (39–41), and diacylglycerol levels are significantly increased in NASH (42). A role for PKCδ in apoptosis has not been described. PKCθ has recently been shown to be activated by endoplasmic reticulum stress in liver cells (43) and lipids in vivo (44, 45). Furthermore, PKCθ has also been implicated in apoptosis of Jurkat cells, neuroblastoma cells, and myeloid leukemia cells (46, 47). However, neither its role in mediating lipoapoptosis nor modulating levels/activity of Bcl-2 proteins has been examined.This study addresses the role of Mcl-1 and PKCθ in FFA-induced lipoapoptosis. We identify a pathway that involves PKCθ-dependent proteasomal degradation of Mcl-1. Using inhibitors of various steps along this pathway, along with Mcl-1 mutants that are resistant to proteasomal degradation or Ser¹⁵⁹ phosphorylation, our studies implicate Mcl-1 degradation via a PKCθ-dependent process as a critical step in lipoapoptosis.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.